Crystalline forms of a TLR4 agonist and methods for preparing the same

Abstract

Disclosed are novel crystalline forms of the TLR4 agonist according to Formula (1) which show beneficial properties in terms of stability, hygroscopicity, and solubility, and methods of preparing the same.

Description

[0001] GRAF VON STOSCH

[0002] PATENTANWALTSGESELLSCHAFT MBH

[0003] Unser Zeichen / Our Ref. Datum / Date KU02P003W01 December 29, 2025

[0004] Applicant:

[0005] Kupando GmbH

[0006] Crystalline Forms of a TLR4 Agonist and Methods for preparing the same

[0007] Field of the Invention

[0008] The present invention relates to novel crystalline forms of a TLR4 agonist useful as pharmaceutical agents, to methods for their preparation, and to pharmaceutical compositions including these compounds.

[0009] Background of the Invention

[0010] The most effective way to protect individuals from the insidious threat of many infectious diseases is through vaccination. Effective vaccination requires the use of antigens that can elicit an immune response in the host capable of providing subsequent protection against that particular infectious agent for which the vaccine is specific. Thus, the vaccine antigen must be immunogenic enough to induce a level of immune response - humoral and / or cell-mediated - to be protective in the host.

[0011] The use of adjuvants in vaccines is a well-established method to promote a stronger immune response to weakly immunogenic antigens. In addition, adjuvants may also enhance and potentially broaden the immune response by promoting the immunogenicity of weakly immunogenic antigens.

[0012] Until recently, the development of new human adjuvants was held back by a poor understanding of their mechanisms of action. The field was revolutionized by the discovery of the toll-like receptors (TRLs) which are innate immune receptors that directly or indirectly are responsible for detecting pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways (Kaur A et al.: Toll-like receptor (TLR) agonists as a driving force behind next-generation vaccine adjuvants and cancer therapeutics. CurrOpin in Chem Biol2022, 70:102172). Hundreds of ligands targeting various TLRs have been identified and characterized as vaccine adjuvants. Each TLR has its own specific tissue localization and downstream gene signalling pathways, providing the opportunity to precisely tailor adjuvants with specific immune effects. TLR agonists can be combined with other TLR or alternative adjuvants to create combination adjuvants with synergistic or modulatory effects. This work has important implications not only for the development of vaccines against infectious diseases but also for immune-therapies against cancer, allergy, Alzheimer's disease and drug addiction, to name a few.

[0013] TLR4, the most explored member of the TLR family recognizes lipopolysaccharides (LPS), a component of the outer membrane of bacteria. TLR4 is located on the plasma membrane and is predominantly expressed on myeloid lineage cells, as pDCs and naive B cells do not express TLR4 (Vuere C, Liu Y: A comparative review of toll-like receptor 4 expression and functionality in different animal species. Front Immunol 2014, 5:316). TLR4 recognizes LPS with its coreceptor myeloid differentiation factor-2 (MD-2) and CD14 (Park BS, Lee J-O: Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med 2013, 45:e66). Substituted pyrimido [5,4-b]indoles are known to induce TLR4-dpendent NF-KB activation and IL-6 release (Chan M et al.: Identification of substituted pyrimido [5, 4-b] indoles as selective Toll-like receptor 4 ligands. J. Med Chem 2013, 56:4206-4223). Recent studies have further demonstrated that TLR4, through TRIF-mediated pathways can activate cellular response characterized by polyfunctional CD8+ / CD4+ cells and enhanced CTL activity against both cancers and infectious diseases (Kim WS et al.: Promotion of cellular and humoral immunity against foot-and-mouth disease virus by immunization with virus-like particles encapsulated in Monophosphoryl Lipid A and liposomes. Vaccines2Q2Q, 8:633; Sunay MM etal.: Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice. Vaccine 2019, 37:3902-3910; Reintjens NR et al.: Self-adjuvanting cancer vaccines from conjugation-ready lipid A analogues and synthetic long peptides. J Med Chem 2020, 63: 11691-11706).

[0014] Recent work on TLR4 agonists has focused on the development and evaluation of modified products, such as monophosphoryl lipid A (MPLA) (Kim WS etal. supra) and glucopyranosyl lipid A (GLA) (Sunay MM et al., supra) that are structurally related to LPS, but devoid of high pyrogenicity and maintaining strong immunopotentiating characteristics, thereby increasing their feasibility for clinical applications.W02020 / 186229 A1 discloses a TLR4 agonist (referred to as 2B182C) having the chemical structure (anhydrate) as shown in Formula (1):

[0015]

[0016] Formula (1)

[0017] Nonetheless, there is a continuous need for crystalline forms of the TLR4 agonist according to Formula (1) that are more stable, but still possess a high degree of solubility.

[0018] In view of the above, it is the object of the present invention to provide new crystalline forms of the compound according to Formula (1) that exhibit advantageous properties in terms of stability, solubility, hygroscopicity and mechanical properties. It is also an object of the present invention to provide a method of isolating a crystalline form of the TLR4 agonist as represented by Formula (1), as well as a method of preparing a crystalline form of the TLR4 agonist according to Formula (1) in larger amounts using the isolated crystalline forms as a "seed". Furthermore, it is also an object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of the TLR4 agonist according to Formula (1).

[0019] This object is achieved by means of the subject-matter set out below and in the appended claims.

[0020] Summary of the invention

[0021] The present invention provides new crystalline forms of the TLR4 agonist according to Formula

[0022]

[0023] Formula (1),

[0024] referred to as 2 Bl 82C in WO 2020 / 186229 A1, and methods for their isolation and preparation, respectively. The new crystalline forms of the TLR4 agonist according to Formula (1) can inter alia be distinguished by their distinct X-ray powder diffraction patterns.

[0025] Thus, in one aspect, the present invention provides a crystalline form of the TLR4 agonist according to Formula (1) selected from the group consisting of:

[0026] (i) a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E);

[0027] (ii) a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F); and

[0028] (iii) a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

[0029] In another aspect, the present invention provides a method of obtaining crystalline forms of the TLR4 agonist according to any one of (i) to (iii) as defined above, comprising the step of suspending a starting material comprising at least one crystalline form of a compound according to Formula (1), preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of about 6.9, and 16.9, ± 0.2° 20, and more preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of about 6.9, 13.1, and 16.9, + 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelengthof 1.5406 A, in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, dimethylformamide / isopropylacetate (1:1, v:v), methyl ethyl ketone, dimethylacetamide / acetonitrile (1:1, v:v), dimethylformamide, and 1,4-dioxane, and obtaining a crystalline form of a compound according to Formula (1 ) by precipitation.

[0030] In particular, the present invention provides a method of obtaining crystalline forms of the TLR4 agonist according to Formula (1 ),

[0031]

[0032] Formula (1)

[0033] comprising the step of suspending a starting material comprising a crystalline form of a compound according to Formula (1), having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 26 of 6.9, and 16.9, ± 0.2° 29 (Pattern A, or Pattern I), and more preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, 13.1, and 16.9, ± 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern A), in a solvent, and obtaining a crystalline form of a compound according to Formula (1), which is different from the crystalline form of the starting material, by precipitation, wherein

[0034] (i) the solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), dimethylformamide / isopropyl acetate (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, methyl ethyl ketone, dimethylacetamide / acetonitrile (1:1, v:v), to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E); or

[0035] (ii) the solvent is selected from 1,4-dioxane to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F).In a further aspect, the present invention provides a method of obtaining a crystalline form of the TLR4 agonist represented by Formula (1):

[0036]

[0037] Formula (1),

[0038] wherein the method comprises the step of competitively equilibrating a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20 (Pattern E), and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20 (Pattern F), in a solvent selected from acetonitrile, acetonitrile / water (1:1, v:v), tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), acetone, and dimethylformamide / isopropylacetate (1:1, v:v) to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

[0039] In still a further aspect, the present invention provides a method for scaled-up preparation of a crystalline form of the TLR4 agonist according to any one of (i) to (iii), as defined above, wherein the method comprises suspending a starting material comprising at least one crystalline form of a compound according to Formula (1), preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, and 16.9 ± 0.2° 20 (Pattern I, Pattern A), and more preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, 13.1, and 16.9 ± 0.2° 20 as measured by X- ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern A), in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, dimethylformamide / isopropylacetate (1:1,v:v), and 1,4-dioxane, in presence of a small amount of a crystalline form of the TLR4 agonist to be prepared as a seed.

[0040] In a further aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one crystalline form of the TLR4 agonist according to any one of (i) to (iii) as defined above, or of at least one crystalline form of the TLR4 agonist as isolated / obtained by a method as defined herein, or of at least one crystalline form of the TLR4 agonist as prepared by a method as defined herein, and at least one pharmaceutically acceptable excipient, optionally in combination with at least further active ingredient.

[0041] In a still further aspect, the present invention provides a crystalline form of the TLR4 agonist according to any one of (i) to (iii) as defined herein, or a crystalline form of the TLR4 agonist as isolated / obtained by a method as defined herein, or a crystalline form of the TLR4 agonist as prepared by a method as described herein for use as an adjuvant or anti-cancer drug.

[0042] In still another aspect, the present invention provides a pharmaceutical composition comprising at least one crystalline form of the TLR4 agonist according to the present invention for use as a vaccine or an anti-cancer medicament.Brief description of the Figures

[0043] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0044] Figure 1 shows an XRPD pattern of compound 2B182C Pattern E, sample ID FRO335O-3- SU4-TC-ACN-300mg;

[0045] Figure 2 shows a DSC thermogram of compound 2B182C Pattern E, sample ID FRO335O- 3-SU4-TC-ACN-300mg;

[0046] Figure 3 shows a TGA thermogram of compound 2B182C Pattern E, sample ID FRO335O- 3-SU4-TC-ACN-300mg;

[0047] Figure 4 shows a1H-NMR spectrum of compound 2B182C Pattern E, sample ID FRO335O- 3-SU4-TC-ACN-300mg;

[0048] Figure 5 shows a SEM photograph of compound 2B182C Pattern E, sample ID FRO335O- 3-SU4-TC-ACN-300mg;

[0049] Figure 6 shows an XRPD pattern of compound 2B182C Pattern F, sample ID FRO335O-3- SU5-TC-dioxane-300mg;

[0050] Figure 7 shows a DSC thermogram of compound 2B182C Pattern F, sample ID FRO335O- 3 -S U 5 -TC-d i oxa ne-3 OOmg;

[0051] Figure 8 shows a TGA thermogram of compound 2B182C Pattern F, sample ID FRO335O- 3-SU5-TC-dioxane-300mg;

[0052] Figure 9 shows a ’H-NMR spectrum of compound 2B182C Pattern F, sample ID FRO335O- 3-SU5-TC-dioxane-300mg;

[0053] Figure 10 shows a SEM photograph of compound 2B182C Pattern F, sample ID FRO335O- 3-SU5-TC-dioxane-300mg;Figure 11 shows an XRPD pattern of compound 2 B 182C Pattern L, sample ID FRO335O-3- SU14-EQ-THF-200mg;

[0054] Figure 12 shows a DSC thermogram of compound 2B182C Pattern L, sample ID FR03350- 3-SU14-EQ-THF-200mg;

[0055] Figure 13 shows a TGA thermogram of compound 2 B 182C Pattern L, sample ID FRO335O- 3-SU14-EQ-THF-200mg;

[0056] Figure 14 shows a1H-NMR spectrum of compound 2B182C Pattern L, sample ID FR03350- 3-SU14-EQ-THF-200mg;

[0057] Figure 15 shows a SEM photograph of compound 2B182C Pattern L, sample ID FRO335O- 3-SU14-EQ-THF-200mg;

[0058] Figure 16 shows an XRPD pattern of compound 2B182C Pattern A, batch PJ02411-37-FP- DRY;

[0059] Figure 17 shows a DSC thermogram of compound 2B182C Pattern A, batch PJ02411 -37- FP-DRY;

[0060] Figure 18 shows a TGA thermogram of compound 2B182C Pattern A, batch PJ02411-37- FP-DRY;

[0061] Figure 19 shows a1H-NMR spectrum of compound 2B182C Pattern A, batch PJ02411-37- FP-DRY;

[0062] Figure 20 shows a PLM photograph of compound 2B182C Pattern A, batch PJ02411 -37-FP- DRY;

[0063] Figure 21 shows an XRPD overlay of polymorphs and pseudopolymorphs isolated in the screening experiments, wherein Fig. 21 A shows Patterns A-F, and Fig. 21 B shows Patterns G-L;Figure 22 shows an XRPD pattern of 2B182C Pattern B, sample ID FRO335O-3-SU1-TC- MeOH-300mg;

[0064] Figure 23 shows an XRPD pattern of 2B182C Pattern C, sample ID FRO335O-3-SU2-TC- EtOH-300mg;

[0065] Figure 24 shows an XRPD pattern of 2B182C Pattern I, batch CH1857-2-7A;

[0066] Figure 25 shows a DSC thermogram of compound 2B182C Pattern I, batch CH1857-2-7A;

[0067] Figure 26 shows an XRPD pattern of 2B182C Pattern D, sample ID FR03350-3-SU3-TC- acetone-300mg;

[0068] Figure 27 shows an XRPD pattern of an amorphous form of 2B182C, sample ID FRO335O- 2-FE1-DCM.

[0069] Detailed description of the invention

[0070] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0071] Definitions

[0072] The term "toll-like receptor agonist" (TLR agonist) refers to a molecule that binds to a TLR. Synthetic TLR agonists are chemical compounds that are designed to bind to a TLR and activate the receptor.

[0073] Throughout this specification and the claims which follow, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", will be understood toimply the inclusion of a stated member, integer or step but not the exclusion of any other nonstated member, integer or step. The term "consist of" is a particular embodiment of the term "comprise", wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term "comprise" encompasses the term "consist of". The term "comprising" thus encompasses "including" as well as "consisting" e.g., a composition "comprising" X may consist exclusively of X or may include something additional e.g., X + Y.

[0074] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0075] The word "substantially" does not exclude "completely"; e.g. a composition which is "substantially free" from Y may be completely free from Y. Similarly, the word "substantially" does not exclude "exactly"; e.g. a diagram which "substantially" corresponds to an indicated figure may exactly correspond to said figure. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[0076] The term "about" in relation to a numerical value x means x ± 10%, for example, x + 5%, or x ± 7%, or x ± 10%, or x ± 12%, or x ± 15%, or x ± 20%.

[0077] Polymorphs of the TLR4 agonist according to Formula (1)

[0078] In extensive studies, the present inventors identified a number of crystalline forms, also referred as "polymorphs", of a TLR4 agonist according to Formula (1) above having beneficial physical properties. In particular, the present inventors identified several crystalline forms of the TLR4 agonist according to Formula (1), including polymorphs referred to as Pattern E, Pattern F and Pattern L, respectively, having improved physical properties in terms of solubility, hygroscopicity and stability compared to other isolated crystalline forms of the TLR4 agonist according to Formula (1). The isolated crystalline forms of the TLR4 agonist according to Formula (1) wereparticularly characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), and ’H-NMR spectrometry.

[0079] Polymorph Pattern E

[0080] In a first embodiment, the present invention provides a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. Preferably this crystalline form of the TLR4 agonist according to Formula (1) also comprises characteristic peaks at 6.9°, 9.2°, and 13.1° ± 0.2° 20, preferably ± 0.1° 20, so that a preferred crystalline form of the TLR4 agonist according to Formula (1) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

[0081] In a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1 ) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.56, 13.76, and 17.36 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. Preferably, this crystalline form of the TLR4 agonist according to Formula (1) also comprises characteristic peaks at 6.90°, 9.16°, 13.12° ± 0.1° 20, preferably ± 0.05° 20, so that a preferred crystalline form of the TLR4 agonist according to Formula (1) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

[0082] Thus, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56, 13.76, and 17.36 ± 0.1° 20, preferably ± 0.05° 20, and preferably having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° ± 0.1 ° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, has an X-ray powder diffraction pattern substantially as shown in Figure 1 as " Pattern E".

[0083] The crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably +0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1 ° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), may be further characterized by its specific melting and recrystallization peaks in differential scanning calorimetry (DSC), its relative change in mass, as e.g. determined by thermal gravimetric analysis (TGA), and its specific1H- NMR spectrum.

[0084] Thus, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1 ° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), may be characterized by a melting peak at Tonsetof 246.9°C + 2°C, preferably ± 1°C, and a recrystallization peak at Tonsetof 251.4°C ± 2°C, preferably + 1°C, as measured by differential scanning calorimetry (DSC). A representative DSC thermogram of polymorph Pattern E is shown in Figure 2. Accordingly, the crystalline form of theTLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56, 13.76, and 17.36 ± 0.2° 20, preferably + 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), may be characterized by a DSC thermogram substantially as shown in Figure 2.

[0085] Moreover, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1 ° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), may be characterized by a relative change in mass of about 0.6% at about 230°C, as determined by thermal gravimetric analysis (TGA). A representative TGA thermogram of polymorph Pattern E is shown in Figure 3. Accordingly, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, and more preferably having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 2θ, preferably + 0.05° 2θ, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 Å (Pattern E), may be characterized by a TGA thermogram substantially as shown in Figure 3.Moreover, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 + 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1 ° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), may be characterized by a ’H-NMR spectrum substantially as shown in Figure 4.

[0086] As shown in the Examples below, a crystalline form of the TLR4 agonist described above (referred to as " Pattern E") shows beneficial physical properties, in particular with respect to stability, hygroscopicity, and solubility.

[0087] Polymorph Pattern F

[0088] In a second embodiment, the present invention provides a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. Preferably this crystalline form of the TLR4 agonist according to Formula (1 ) also comprises characteristic peaks at 7.3°, 9.7°, 19.1, 20.5 and 21.4 + 0.2° 20, preferably ± 0.1° 20, so that a preferred crystalline form of the TLR4 agonist according to Formula (1 ) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

[0089] In a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.73, 10.88, and 15.49 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. Preferably this crystalline form of theTLR4 agonist according to Formula (1) also comprises characteristic peaks at 7.32°, 9.71°, 19.05, 20.50, and 21.38± 0.1° 20, preferably ± 0.05° 20, so that a preferred crystalline form of the TLR4 agonist according to Formula (1) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.Thus, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73, 10.88, and 15.49 ± 0.1° 20, preferably + 0.05° 20, and preferably having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 7.32°, 9.71 °, 10.88°, 15.49°, 19.05, 20.50, and 21.38 + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, has an X-ray powder diffraction pattern substantially as shown in Figure 6 as Pattern F.

[0090] The crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 10.88°, and 15.49° + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), may be further characterized by its specific recrystallization peak in differential scanning calorimetry (DSC), its relative change in mass as e.g. determined by thermal gravimetric analysis (TGA), and its specific ’H-NMR spectrum.

[0091] Thus, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), may be characterized by a recrystallization peak at Tonset of 195.3°C ± 2°C, preferably ± 1°C, as measured by differential scanning calorimetry. A representative DSC thermogram of polymorph Pattern F is shown in Figure 7. Accordingly, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably ± 0.1 ° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1 ° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), may be characterized by a DSC thermogram substantially as shown in Figure 7.

[0092] Moreover, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably + 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 20, preferably + 0.05°29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), may be characterized by a relative change in mass of about 0.6% at 175°C, and of about 0.6% from 175°C to 240°C, as determined by thermal gravimetric analysis. A representative TGA thermogram of polymorph Pattern F is shown in Figure 8. Accordingly, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, preferably ± 0.1° 29, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), may be characterized by a TGA thermogram substantially as shown in Figure 8.

[0093] Moreover, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 29, preferably ± 0.1 ° 29, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1 ° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), may be characterized by a1H-NMR spectrum substantially as shown in Figure 9.

[0094] As shown in the Examples below, a crystalline form of theTLR4 agonist described above (referred to as Pattern F) shows beneficial physical properties, in particular with respect to stability, hygroscopicity, and solubility.

[0095] Polymorph Pattern L

[0096] In a third embodiment, the present invention provides a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 + 0.2° 29, preferably ± 0.1° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. Preferably this crystalline form of the TLR4 agonist according to Formula (1) also comprises characteristic peaks at 15.6, 21.5, and 22.8 ± 0.2° 29, preferably ± 0.1° 29, so that a preferred crystalline form of the TLR4 agonist according to Formula (1) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 + 0.2° 29, preferably ± 0.1° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.In a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 + 0.1° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. Preferably this crystalline form of the TLR4 agonist according to Formula (1) also comprises characteristic peaks at 15.58, 21.55, and 22.82 ± 0.1° 20, preferably ± 0.05° 20, so that a preferred crystalline form of the TLR4 agonist according to Formula (1 ) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

[0097] Thus, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 20, preferably ± 0.05° 20, and preferably having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 + 0.1° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, has an X-ray powder diffraction pattern substantially as shown in Figure 11 as Pattern L.

[0098] The crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 + 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), may be further characterized by its specific recrystallization peak in differential scanning calorimetry (DSC), its relative change in mass determined by thermal gravimetric analysis (TGA), and its specific1H-NMR spectrum.

[0099] Thus, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 29, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), may be characterized by a recrystallization peak at Tonset of 212.8°C + 2°C, preferably + 1°C, as measured by differential scanning calorimetry. A representative DSC thermogram of polymorph Pattern L is shown in Figure 12. Accordingly, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5,10.7, and 19.4 ± 0.2° 29, preferably + 0.1 ° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 29, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), may be characterized by a DSC thermogram substantially as shown in Figure 12.

[0100] Moreover, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 29, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 20, preferably + 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), may be characterized by a relative change in mass of about 0.8% at 180°C as determined by thermal gravimetric analysis. A representative TGA thermogram of polymorph Pattern L is shown in Figure 13. Accordingly, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 20, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 + 0.1° 20, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), may be characterized by a TGA thermogram substantially as shown in Figure 13.

[0101] Moreover, in a particular embodiment, the crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 2θ, or particularly having an X-ray powder diffraction pattern comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), may be characterized by a1H-NMR spectrum substantially as shown in Figure 14.

[0102] As shown in the Examples below, a crystalline form of the TLR4 agonist described above (referred to as Pattern L) shows particularly beneficial physical properties, in particular with respect to stability, hygroscopicity, solubility, as well as excellent mechanical properties.

[0103] Methods of obtaining the polymorphs of the TLR4 agonist according to Formula (1)

[0104] The crystalline forms of the TLR4 agonist according to Formula (1) as described above, also referred to as "polymorphs" of 2B182C, having(i) an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 13.8°, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (referred to as Pattern E); or

[0105] (ii) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (referred to as Pattern F); or

[0106] (iii) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (referred to as Pattern L),

[0107] may be obtained by using a starting material comprising at least one anhydrous form of a compound according to Formula (1) which is different from the crystalline forms or polymorphs according to (i) to (iii).

[0108] In an embodiment, the starting material comprising at least one anhydrous form of a compound according to Formula (1 ) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.9, and 16.9, ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

[0109] In a particular embodiment, the starting material comprising at least one anhydrous form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.9, and 16.9, ± 0.2° 20, preferably ± 0.1° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, may be characterized by an XRPD pattern substantially as shown in Figure 24 (referred to as Pattern I).

[0110] Moreover, the starting material comprising at least one anhydrous form of a compound according to Formula (1), may be characterized by a melting peak at Tonset of 260.5 °C ± 2°C, preferably ± 1°C, as measured by differential scanning calorimetry. A representative DSC thermogram of 2B182C Pattern I which may be used as a starting material for obtaining the polymorphs according to the present invention is shown in Figure 25. Accordingly, the starting material (2B182C Pattern I) for obtaining the polymorphs according to the present invention may be characterized by a DSC thermogram substantially as shown in Figure 25.In a preferred embodiment, the starting material comprising at least one anhydrous form of a compound according to Formula (1) and having an X-ray powder diffraction pattern comprising characteristic peaks at 6.9, and 16.9, ± 0.2° 26, preferably ± 0.1° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, is characterized by an X-ray powder diffraction pattern comprising characteristic peaks at 6.9, 13.1, and 16.9, ± 0.2° 26, preferably ± 0.1 ° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (referred to as 2 B 182 C Pattern A).

[0111] In a particular embodiment, the starting material (Pattern A) comprising at least one anhydrous form of a compound according to Formula (1 ) has an X-ray powder diffraction pattern comprising characteristic peaks at 6.91, 13.14, and 16.95, ± 0.1° 26, preferably ± 0.05° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A. For example, the starting material (2B182C Pattern A) for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) may be characterized by an XRPD pattern substantially as shown in Figure 16.

[0112] Moreover, the starting material (2B182C Pattern A) for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) may be characterized by a melting peak at Tonset of 260.7 °C ± 2°C, preferably ± 1°C, as measured by differential scanning calorimetry. A representative DSC thermogram of 2B182C Pattern A is shown in Figure 17. Accordingly, the starting material (2B182C Pattern A) for obtaining the polymorphs according to the present invention may be characterized by a DSC thermogram substantially as shown in Figure 17.

[0113] Furthermore, the starting material (2B182C Pattern A) for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) may be characterized by a relative change in mass of about 0.5% at 170°C, and of about 0.3% from 170°C to 210 °C, as determined by thermal gravimetric analysis. A representative TGA thermogram of 2B182C Pattern A is shown in Figure 18. Accordingly, the starting material (2B182C Pattern A) for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) may be characterized by a TGA thermogram substantially as shown in Figure 18.

[0114] Further, the starting material (2B182C Pattern A) used for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) may be characterized by1H-NMR spectrum substantially as shown in Figure 19. It is noted that the starting material (referred to as " PatternA") which may be used for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) is actually a mixture of polymorphic forms of the compound according to Formula (1), in particular a mixture of polymorphs identified as Pattern I and Pattern D, which may be characterized by XRPD patterns substantially as shown in Figures 24 (for Pattern I) and 26 (for Pattern D), respectively.

[0115] The present inventors found that the starting material (e.g. Pattern I or Pattern A) which may be used for obtaining the polymorphs according to the present invention (Pattern E, Pattern F, Pattern L) may be obtained by a process involving the synthesis of several intermediate compounds as described below.

[0116] The starting compound for the synthesis of 2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-(5,4-b)indol-2-yl)thio)-N-cyclohexylacetamide according to Formula (1), which is used as a starting material for obtaining the polymorphs according to the present invention, is ethyl-3-amino-5-bromo-1 H-indole-2 -carboxylate represented by Formula (2):

[0117]

[0118] as described in M. Chan etal., Structure-Activity Relationship Studies of Pyrimido[5,4-Z?]lindoles as Selective Toll-Like Receptor 4 Ligands, / . Med. Chem., 2017, 60, 9142-9161, the content of which is incorporated herein by reference.

[0119] Starting from a compound according to Formula (2), a compound according to Formula (1) may be obtained by a (novel) synthesis method comprises the steps of:

[0120] i) reacting ethyl-3-amino-5-bromo-1 H-indole-2 -carboxylate according to Formula (2) with methyl iodide to obtain ethyl-1-methyl-3-amino-5-bromo-1 H-indole-2 - carboxylate according to Formula (3):

[0121]

[0122] ii) reacting ethyl-1-methyl-3-amino-5-bromo-1 H-indole-2 -carboxylate obtained in step i) with 2-furanboronic acid pinacol ester to obtain ethyl-1 -methyl-3-amino- 5-(2-fury l)-1 H-indole-2 -carboxylate according to Formula (4):

[0123]

[0124] iii) reacting ethyl-1 -methyl-3-amino-5-(2-furyl)-1 H-indole-2-carboxylate obtained in step ii) with phenyl isothiocyanate to obtain ethyl-1 -methyl-5-(2-furyl)-3-(3- phenylthioureido)amino-1 H-indole-2 -carboxylate according to Formula (5):

[0125]

[0126] iv) reacting ethyl 1-methyl-5-(2-furyl)-3-(3-phenylthioureido)amino-1 H-indole-2 - carboxylate obtained in step iii) with sodium ethoxide to obtain 8-(2-furyl)-5- methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4-b]-indol-4(5H)-one according to Formula (6):

[0127]

[0128] (6); and

[0129] v) reacting 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4- b]-indol-4(5H)-one obtained in step iv) with 2-chloro-N-cyclohexylacetamide to obtain 2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-(5,4- b)indol-2-yl)thio)-N-cyclohexylacetamide according to Formula (1):

[0130]

[0131] In the above described synthesis method, the reaction in step i), i.e. reacting ethyl-3-amino-5-bromo-1 H-indole-2 -carboxylate according to Formula (2) with methyl iodide, is preferably conducted in dimethylformamide in the presence of sodium hydride. Preferably, the methylated product obtained in step i) (i.e. ethyl-1 -methyl-3-amino-5-bromo-1 H-indole-2 -carboxylate according to Formula (3)) is extracted with a suitable extraction solvent such as ethyl acetate, and dried, e.g. over MgSO4, before it is subjected to step ii).

[0132] In the above described synthesis method, the reaction in step ii), i.e. reacting ethyl-1 -methyl-3-amino-5-bromo-1 H-indole-2 -carboxylate obtained in step i) with 2-furanboronic acid pinacol ester, is preferably conducted in dimethoxyethane. Preferably, step ii) is accomplished in the presence of sodium carbonate and water. Further, the reaction in step ii) is preferably conducted in the presence of a catalyst. Preferably, a transition metal catalyst such as [1,1 '-bis(diphenylphosphino)ferrocene] dichloropalladium (II) is used in step ii). Preferably, the product obtained in step ii) is extracted with a suitable extraction solvent such as ethyl acetate. Moreover a metal scavenger such as SiliaMetS® Di mercaptotri azine may be used to remove the catalyst from the organic layer. Further, crystallization of the intermediate product (i.e. ethyl-1 - methyl-3-amino-5-(2-furyl)-1 H-indole-2-carboxylate according to Formula (4)) may be facilitated by a crystallising agent, such as isopropanol.

[0133] In the above described synthesis method, the reaction in step iii), i.e. reacting ethyl-1 -methyl-3-amino-5-(2-furyl)-1 H-indole-2 -carboxylate obtained in step ii) with phenyl isothiocyanate, is preferably conducted in acetonitrile. Preferably, a precipitation agent, such as isopropanol, is added after the reaction to allow precipitation of the intermediate product ethyl-1 -methyl-5-(2- furyl)-3-(3-phenylthioureido)amino-1 H-indole-2 -carboxylate according to Formula (5).

[0134] In the above described synthesis method, the reaction in step iv), i.e. reacting ethyl-1 -methyl-5-(2-furyl)-3-(3-phenylthioureido)amino-1 H-indole-2 -carboxylate obtained in step iii) with sodium ethoxide, is preferably conducted in EtOH, more preferably in anhydrous EtOH. Preferably, aprecipitation agent, such as hydrochloric acid, is added after the reaction to facilitate precipitation of the intermediate product 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4-b]-indol-4(5H)-one according to Formula (6).

[0135] In the above described synthesis method, the reaction in step v), i.e. reacting 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4-b]-indol-4(5H)-one obtained in step iv) with 2-chloro-N-cyclohexylacetamide, is preferably conducted in dimethylformamide. Preferably, step v) is accomplished in the presence of triethylamine. Preferably, a precipitation agent, such as isopropanol, is added after the reaction to allow precipitation of the intermediate. Further, recrystallization of the final product (2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-(5,4-b)indol-2-yl)thio)-N-cyclohexylacetamide according to Formula (1)) may be accomplished by using a recrystallising agent, such as DMSO-isopropanol.

[0136] Accordingly, the present disclosure also provides an (intermediate) compound, ethyl-1-methyl-3-amino-5-bromo-1 H-indole-2 -carboxylate, according to Formula (3):

[0137]

[0138] (3).

[0139] Further, the present disclosure provides an (intermediate) compound, ethyl 1 -methyl-3-amino- 5-(2-furyl)-1 H-indole-2-carboxylate, according to Formula (4):

[0140]

[0141] Further, the present disclosure provides an (intermediate) compound, ethyl 1-methyl-5-(2-furyl)- 3-(3-phenylthioureido)amino-1 H-indole-2 -carboxylate, according to Formula (5):

[0142]

[0143] Further, the present disclosure provides an (intermediate) compound, 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4-b]-indol-4(5H)-one, according to Formula (6):

[0144] o

[0145]

[0146] Using 2B182C Pattern A and 2B182C Pattern I, respectively, having the physicochemical properties described above as a starting material, the present inventors carried out extensive screening experiments and studies thereby identifying a number of crystalline forms of the TLR4 agonist as represented by Formula (1), also referred as "polymorphs" of 2B182 C.

[0147] Thus, in a further aspect, the present invention provides a method of obtaining a crystalline form of the TLR4 agonist as represented by Formula (1 ):

[0148]

[0149] Formula (1),

[0150] as defined above, comprising the step of suspending a starting material comprising at least one crystalline form of a compound according to Formula (1), having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, and 16.9 ± 0.2° 20, preferably ± 0.1 ° 20 (2B182C Pattern I or Pattern A) and more preferably having an X-ray powderdiffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, 13.1, and 16.9 ± 0.2° 20, preferably ± 0.1 ° 29, in particular 6.91, 13.14, and 16.95, ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (2B182C Pattern A), in an appropriate solvent and obtaining a crystalline compound, which is different from the crystalline form used as a starting material, by precipitation.

[0151] Using 2B182C Pattern I or Pattern A (which comprises a mixture of different polymorphic forms of a compound according to Formula (1), i.e. Pattern I and Pattern D) as a starting material, the inventors developed methods to selectively obtain the desired polymorphic patterns, in particular Patterns E, F and L, having improved solubility, hygroscopicity and stability characteristics.

[0152] In the method according to the present invention for obtaining / isolating new crystalline forms of the TLR4 agonist as represented by Formula (1) having improved properties, in particular in respect of solubility, hygroscopicity, and stability (crystalline forms according to Pattern E, F and L, respectively), the solvent may be selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, dimethylformamide / isopropylacetate (1:1, v:v), methyl ethyl ketone, dimethylacetamide / acetonitrile (1:1, v:v) dimethylformamide and 1,4-dioxane. In the method according to the present invention for obtaining / isolating a crystalline form of the TLR4 agonist as represented by Formula (1), the precipitate may be formed by evaporation of the solvent. In a preferred embodiment, the suspension is filtered to obtain the precipitate.

[0153] Isolation of polymorph Pattern E

[0154] The present inventors found that a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 1°C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1 ° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), as described above, can be obtained by suspending a starting material (e.g. 2B182C Pattern A or Pattern I, preferably Pattern A), as characterized above, in particular a starting material comprising at least one crystalline form of a compound according to Formula (1), having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, and 16.9 ± 0.2° 20, preferably ± 0.1° 29 (Pattern I or Pattern A) and more preferably having an X-ray powder diffraction pattern comprising characteristic peaks at areflection angle 29 of 6.9, 13.1, and 16.9 ± 0.2° 29, preferably ± 0.1° 29, in particular 6.91, 13.14, and 16.95, + 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern A), in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), dimethylformamide / isopropyl acetate (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, metyl ethyl ketone, and dimethylacetamide / acetonitrile (1:1, v:v), and obtaining a crystalline form of the obtained compound by precipitation.

[0155] In particular, the present inventors found that a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 29, preferably ± 1°C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 29, preferably + 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), as described above, can be obtained by suspending a starting material (e.g. 2B182C Pattern A or Pattern I, preferably Pattern A), as characterized above, in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), and dimethylformamide / isopropyl acetate (1:1, v:v), and equilibrating the suspension at a temperature of about 25°C for a period of about 2 to 3 weeks.

[0156] Moreover, the present inventors found that, alternatively, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 29, preferably + 1 °C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1 ° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), can be obtained by suspending a starting material, as characterized above (e.g. Pattern A or Pattern I, preferably Pattern A), in a solvent selected from the group consisting of ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, dimethylsulfoxide, and dimethylformamide / isopropyl acetate (1:1, v:v), and equilibrating the suspension at a temperature of about 50°C for a period of about 1 to 2 weeks.

[0157] Moreover, the present inventors found that, according to a further alternative, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 29, preferably + 1 °C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), ascharacterized above, in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), and dimethylformamide / isopropyl acetate (1:1, v:v), and equilibrating the suspension under a temperature cycle between about 5°C and about 50°C, preferably at a heating / cooling rate of about 0.2 °C / min, for about 10 cycles.

[0158] Furthermore, the present inventors found that, according to a still further alternative, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 1 °C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), as characterized above, in a solvent selected from the group consisting of methyl ethyl ketone, tetrahydrofuran, dimethylsulfoxide, and dimethylacetamide / acetonitrile (1:1, v:v) at a temperature of about 50°C, and slow cooling the solution to a temperature of about 5°C to -20°C, preferably at a rate of about 0.1 °C / min.

[0159] Furthermore, the present inventors found that, according to a yet further alternative, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 1 °C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), as characterized above, in a mixture of dimethylacetamide / acetonitrile (1:1, v:v) at a temperature of about 50°C, and fast cooling the solution to a temperature of about 0°C, for example by placing the solution in an ice bath.

[0160] Moreover, the present inventors found that, according to a further alternative, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably + 1°C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), as characterized above, in dimethylsulfoxide and adding acetonitrile (e.g. 4-8 fold amount) as an anti-solvent.The suspension obtained by any of the methods described above for obtaining / isolating a crystalline form of a TLR4 agonist may be filtered to obtain the precipitate of the crystalline form of the compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 + 0.2° 20, preferably ± 1 °C, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E). Preferably, the precipitates are collected by centrifugation filtration through a filter having an appropriate pore size. For example, a 0.45 pm nylon membrane filter may be used to collect the precipitates. Centrifugation may, for example, be performed at about 14,000 rpm.

[0161] Isolation of polymorph Pattern F

[0162] Further, the present inventors found that a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), as defined above, can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), i.e. a starting material comprising at least one crystalline form of a compound according to Formula (1), having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, and 16.9 ± 0.2° 20, preferably ± 0.1 ° 20 (2B182C Pattern I or Pattern A) and more preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, 13.1, and 16.9 ± 0.2° 20, preferably ± 0.1 ° 20, in particular 6.91, 13.14, and 16.95, ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern A), in a solvent selected from 1,4-dioxane, and obtaining a crystalline form of the obtained compound by precipitation.

[0163] In particular, the present inventors found that a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably + 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), as defined above, can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), ascharacterized above, in 1,4-dioxane and equilibrating the suspension at a temperature of about 25 °C for about two weeks.

[0164] Moreover, the present inventors found that, alternatively, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20, preferably + 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F) can be obtained by suspending a starting material (e.g. Pattern A or Pattern I, preferably Pattern A), as characterized above, in 1,4-dioxane and equilibrating the suspension at a temperature of about 50°C for about one week.

[0165] Moreover, the present inventors found that, in a further alternative, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° + 0.1° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F) can be obtained by suspending the starting material (e.g. Pattern A or Pattern I, preferably Pattern A), as characterized above, in 1,4-dioxane, and equilibrating the suspension under a temperature cycle between about 5°C and about 50°C, preferably at a heating / cooling rate of about 0.2 °C / min, preferably for about 10 cycles.

[0166] The suspension obtained by any of the methods described above for obtaining / isolating a polymorph of Pattern F may be filtered to obtain the precipitate of the crystalline form of the compound according to Formula (1). Preferably, the precipitates are collected by centrifugation filtration through a filter having an appropriate pore size. For example, a 0.45 pm nylon membrane filter may be used to collect the precipitates. Centrifugation may, for example, be performed at about 14,000 rpm.

[0167] Isolation of polymorph Pattern L

[0168] Furthermore, the present inventors found that a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 + 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1 ° 20, preferably ± 0.05° 20, as measured by X-ray powder diffractionusing an X-ray wavelength of 1.5406 A (Pattern I), as defined above, can be obtained by competitively equilibrating a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 26, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 26, preferably ± 0.05° 20 (Pattern E), e.g. obtained from Pattern A or Pattern I, preferably Pattern A, as a starting material, as described above, and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, 10.9, and 15.5 ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 29, preferably ± 0.05° 29 (Pattern F), e.g. obtained from Pattern A or Pattern I, preferably Pattern A, as a starting material, as described above, in a solvent selected from acetonitrile, acetonitrile / water (1:1, v:v), tetrahydrofuran, acetone, dimethylformamide / isopropylacetate (1:1, v:v), and tetrahydrofuran / water (1:1, v:v).

[0169] In particular, the present inventors found that a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 29, preferably + 0.1° 29, or particularly comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1 ° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), as defined above, can be obtained by competitively equilibrating a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 + 0.2° 29, preferably ± 0.1° 2θ, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), e.g. obtained from Pattern A or Pattern I, preferably Pattern A, as a starting material, as described above, and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° + 0.1° 29, preferably + 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), e.g. obtained from Pattern A or Pattern I, preferably Pattern A, as a starting material, as described above, in a solvent selected from acetonitrile, and acetonitrile / water (1:1, v:v), at a temperature of about 25°C. To obtain Pattern L by competitive equilibration at about 25°C, equilibration is preferably performed over a period of at least about 25 days.Moreover, the present inventors found that, alternatively, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 + 0.2° 29, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L) can be obtained by competitively equilibrating a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 29, preferably + 0.1° 29, or particularly comprising characteristic peaks at 6.56°, 13.76°, and 17.36° ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E), e.g. obtained from Pattern A or Pattern I, preferably Pattern A, as a starting material, as described above, and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, preferably ± 0.1 ° 29, or particularly comprising characteristic peaks at 6.73°, 10.88°, and 15.49° ± 0.1° 29, preferably + 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), e.g. obtained from Pattern A or Pattern I, preferably Pattern A, as a starting material, as described above, in a solvent selected from acetonitrile, tetrahydrofuran, acetonitrile / water (1:1, v:v), acetone, dimethylformamide / isopropyl acetate (1:1, v:v), and tetrahydrofuran / water (1:1, v:v) at a temperature of about 50°C. In a preferred embodiment, Pattern L may be obtained from patters E and F by competitive equilibration at about 50°C in a solvent selected from acetonitrile, tetrahydrofuran, and acetonitrile / water (1:1, v:v).

[0170] Moreover, the present inventors found that, alternatively, a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 29, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.46, 10.73, and 19.40 ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L) can be obtained by water activity experiments, as described below, using tetrahydrofuran / water (1:1, v:v) as a solvent.

[0171] The suspension obtained by any of the methods described above for obtaining / isolating a polymorph of Pattern L may be filtered to obtain the precipitate of the crystalline form of the compound according to Formula (1). Preferably, the precipitates are collected by centrifugation filtration through a filter having an appropriate pore size. For example, a 0.45 pm nylonmembrane filter may be used to collect the precipitates. Centrifugation may, for example, be performed at 14,000 rpm.

[0172] Methods of scaled-up preparation of crystalline forms of the TLR4 agonist 2B182C

[0173] The crystalline forms of the TLR4 agonist according to the present invention represented by Formula (1) having the patterns E, F and L as described above which have been obtained / isolated by methods as described above, may be used in methods of preparing crystalline forms of the TLR4 agonist according to Formula (1) in larger amounts, wherein the obtained / isolated crystalline forms of the TLR4 agonist according to Formula (1) (in particular Pattern E, Pattern F and patter L, respectively) are used as a seed.

[0174] Thus, in a further aspect, the present invention provides a method of preparing larger amounts of a crystalline form of the TLR4 agonist according to Formula (1):

[0175]

[0176] Formula (1),

[0177] wherein the method comprises suspending a starting material comprising at least one crystalline form of a compound according to Formula (1), preferably having an X-ray powder diffraction pattern having characteristic peaks at a reflection angle 20 of about 6.9, and 16.9 + 0.2° 20, preferably ± 0.1° 20 (i.e. Pattern I or Pattern A), and preferably having an X-ray powder diffraction pattern having characteristic peaks at a reflection angle 20 of about 6.9, 13.1 and 16.9 ± 0.2° 20, preferably ± 0.1° 20, and particularly having an X-ray powder diffraction pattern having characteristic peaks at a reflection angle 20 of about 6.91, 13.14 and 16.95 ± 0.1° 20, preferably ± 0.05° 20, e.g. a compound having an X-ray powder diffraction pattern substantially as shown in Figure 15 as Pattern A, in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, dimethylformamide / isopropyl acetate (1:1, v:v), and 1,4-dioxane, in presence of a small amount of a crystalline form of the TLR4 agonist to be prepared, in particular a compound having the pattern E, F, or L which has been obtained as described above, as a seed.Scaled-up preparation of polymorph Patern E

[0178] In particular, the present inventors found that a larger amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° + 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Patern E), can be obtained by suspending a starting material comprising at least one compound according to Formula (1), preferably having an X-ray powder diffraction patern comprising characteristic peaks at a reflection angle 20 of about 6.9, and 16.9 + 0.2° 20, preferably ± 0.1° 20, (Patern I or Patern A) and preferably having an X-ray powder diffraction patern comprising characteristic peaks at a reflection angle 20 of about 6.9, 13.1, and 16.9 + 0.2° 20, preferably ± 0.1° 20 (Patern A) in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide and dimethylformamide / isopropyl acetate (1:1, v:v), in the presence of a small amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably + 0.1 ° 20, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably + 0.1° 20, or particularly comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° ± 0.1° 20, preferably ± 0.05° 20 (Patern E) as a seed.

[0179] In a preferred embodiment, the solvent used in the inventive method for preparing a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably + 0.1° 20, or particularly comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° + 0.1° 20, preferably ± 0.05° 20 (Patern E) is acetonitrile.

[0180] In the inventive method for preparing a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1° 20, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristicpeaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° ± 0.1° 2θ, preferably ± 0.05° 20 (Patern E), the suspension is preferably equilibrated using a temperature cycle between about 5°C to about 50°C using a moderate heating / cooling rate. For example, the suspension may be subjected to a temperature cycle between about 5°C to about 50°C at a heating / cooling rate of about 0.1 °C / min.

[0181] In the inventive method for preparing a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction patern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably ± 0.1029, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° ± 0.1 ° 20, preferably ± 0.05° 20 (Patern E), the ratio of the starting material (e.g. Patern A or Patern I, preferably Pattern A) to the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably + 0.1° 20, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, preferably + 0.1° 20, or particularly comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° ± 0.1° 20, preferably ± 0.05° 20 (Patern E) added as a seed may be in a range of about 100:0.1 to about 100:10. Preferably, the ratio of the starting material (e.g. Pattern A or Patern I, preferably Patern A) to the "seed" (Patern E) may be in a range of about 100:0.5 to about 100:5. More preferably, the ratio of the starting material (e.g. Patern A or Patern I, preferably Pattern A) to the "seed" (Patern E) may be in a range of about 100:1 to about 100:3. More preferably, the ratio of the starting material (e.g. Patern A or Patern I, preferably Patern A) to the "seed" (Patern E) may be about 100:1 to about 100:2, most preferably about 100:1 to about 100:1.5.

[0182] In the method according to the present invention, the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 29, preferably + 0.1 ° 29, and preferably comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 29, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.56°, 6.90°, 9.16°, 13.12°, 13.76°, and 17.36° ± 0.1° 29, preferably ± 0.05° 20 (Patern E) is preferably obtained by filtrating the suspension through a filter having an appropriate pore size to obtain the precipitated crystalline form of the compound according to Patern E. For example, a 0.45 pm nylon membrane filter may be used to collect the precipitates. The precipitate may be collected by centrifugation filtration. Centrifugation filtration may be performed at a rate and for a time which is sufficient to obtain the precipitate. Centrifugationfiltration may, for example, be performed at about 14,000 rpm for about 10 minutes. If necessary, the obtained solid may be dried at ambient condition (about 25°C, 20% RH).

[0183] Scaled-up preparation of polymorph Pattern F

[0184] Moreover, the present inventors found that a larger amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably ± 0.1° 20, and preferably comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 20, preferably + 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F) can be obtained by suspending in 1,4-dioxane a starting material comprising at least one crystalline form of a compound according to Formula (1), preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of about 6.9, and 16.9 ± 0.2° 20, preferably ± 0.1 ° 20 (Pattern A or Pattern I), and preferably having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 2θ of about 6.9, 13.1, and 16.9 ± 0.2° 2θ, preferably ± 0.1° 2θ (Pattern A), in the presence of a small amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably + 0.1° 20, and preferably comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 + 0.1° 20, preferably + 0.05° 20 (Pattern F) as a seed.

[0185] In the inventive method for preparing a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably ± 0.1 ° 20, and preferably comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 ± 0.1° 20, preferably + 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), the suspension is preferably equilibrated using a temperature cycle between about 5°C to about 50°C using a moderate heating / cooling rate. For example, the suspension may be subjected to the temperature cycle between about 5°C to about 50°C at a heating / cooling rate of about 0.1 °C / min.In the inventive method for preparing a crystalline form of the compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, preferably ± 0.1029, and preferably comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 29, preferably ± 0.1 ° 29, or particularly comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 + 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, preferably ± 0.1° 29, and preferably comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 29, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 ± 0.1° 29, preferably ± 0.05° 29 (Pattern F) added as a seed may be in a range of about 100:0.1 to about 100:10. Preferably, the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the seed (Pattern F) may be in a range of about 100:0.5 to about 100:5. More preferably, the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the seed (Pattern F) may be in a range of about 100:1 to about 100:3. More preferably, the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the seed (Pattern F) may be about 100:1 to about 100:2, most preferably about 100:1 to about 100:1.5.

[0186] In the inventive method, the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, preferably + 0.1° 29, and preferably comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 29, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.73°, 7.32°, 9.71°, 10.88°, 15.49°, 19.05, 20.50, and 21.38 ± 0.1° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F), is preferably obtained by filtrating the suspension through a filter having an appropriate pore size to obtain the precipitated crystalline form of the compound according to Pattern F. For example, a 0.45 pm nylon membrane filter may be used to collect the precipitate. Preferably, the precipitate is collected by centrifugation filtration. Centrifugation filtration may be performed at a rate and for a time which is sufficient to obtain the precipitate. Centrifugation filtration may, for example, be performed at about 14,000 rpm for about 10 minutes. If necessary, the obtained solid may be dried at ambient condition (about 25°C, 20% RH).Scaled-up preparation of polymorph Patern L

[0187] Moreover, the present inventors found that a larger amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 20, and preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Patern L) can be obtained by suspending a starting material comprising at least one crystalline form of a compound according to Formula (1 ), preferably having an X-ray powder diffraction patern comprising characteristic peaks at a reflection angle 20 of about 6.9, and 16.9 ± 0.2° 20, preferably ± 0.1 ° 20 (e.g. Patern A or Patern I), and preferably having an X- ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of about 6.9, 13.1, and 16.9 ± 0.2° 20, preferably ± 0.1° 20 (Pattern A) in tetrahydrofuran, and equilibrating the suspension in the presence of a small amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably ± 0.1° 20, and preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 + 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 + 0.1° 20, preferably ± 0.05° 20 (Patern I) as a seed.

[0188] In the inventive method for preparing a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction patern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably + 0.1 ° 20, and preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 + 0.2° 20, preferably ± 0.1° 20, or particularly comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 ± 0.1° 20, preferably ± 0.05° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Patern L), the suspension is preferably equilibrated at a temperature of about 50°C. Preferably, the suspension is equilibrated under agitation.

[0189] In the inventive method for preparing a crystalline form of the compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably + 0.1° 20, and preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 + 0.2° 20, preferably ± 0.1° 20, or particularly comprisingcharacteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 + 0.1 ° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks 6.5, 10.7, and 19.4 ± 0.2° 29, preferably ± 0.1° 29, and preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 + 0.2° 29, preferably + 0.1 ° 29, or particularly comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 ± 0.1 ° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L) added as a seed may be in a range of about 100:0.1 to about 100:10. Preferably, the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the seed (Pattern L) may be in a range of about 100:0.5 to about 100:5. More preferably, the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the seed (Pattern L) may be in a range of about 100:1 to about 100:3. More preferably, the ratio of the starting material (e.g. Pattern A or Pattern I, preferably Pattern A) to the seed (Pattern L) may be about 100:1 to about 100:2, most preferably about 100:1 to about 100:1.5.

[0190] In the inventive method, the crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 29, preferably ± 0.1° 29, and preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 29, preferably ± 0.1° 29, or particularly comprising characteristic peaks at 6.46, 10.73, 15.58, 19.40, 21.55, and 22.82 ± 0.1 ° 29, preferably ± 0.05° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), is preferably obtained by filtrating the suspension through a filter having an appropriate pore size to obtain the precipitated crystalline form of the compound according to Pattern L. For example, a 0.45 pm nylon membrane filter may be used to collect the precipitate. Preferably, the precipitate is collected by centrifugation filtration. Centrifugation filtration may be performed at a rate and for a time which is sufficient to obtain the precipitate. For example, centrifugation filtration may be performed at about 14,000 rpm for about 10 minutes. If necessary, the obtained solid may be dried at ambient condition (about 25°C, 20% RH).

[0191] Pharmaceutical compositions

[0192] In a further aspect, the present invention provides a pharmaceutical composition comprising an effective amount of at least one crystalline form of a TLR4 agonist of the present invention or ofat least one crystalline form of a TLR4 agonist as isolated by a method of the present invention, or of at least one crystalline form of a TLR4 agonist as prepared by a method according to the present invention, and a pharmaceutical acceptable excipient. The "effective amount" may be a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be), this being sufficient to show benefit to the individual. The pharmaceutical composition may e.g. be used as a vaccine or an anti-cancer medicament.

[0193] Pharmaceutical compositions of the present invention contain a crystalline form of a TLR4 agonist according to the present invention. In addition to the crystalline form of a TLR4 agonist, the pharmaceutical composition of the present invention may contain one or more excipients. Excipients including disintegrants, glidants, binders, diluents, lubricants, flavoring agents and colorants, may be added to the composition for a variety of purposes.

[0194] Diluents increase the bulk of a solid pharmaceutical composition, and can make a pharmaceutical dosage form containing the composition easier for the patient and caregiver to handle. Diluents for solid compositions include, for example, microcrystalline cellulose, microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates, potassium chloride, powdered cellulose, sodium chloride, sorbitol, and / or talc.

[0195] Solid pharmaceutical compositions that are compacted into a dosage form, such as a tablet, can include excipients whose functions include helping to bind the active ingredient and other excipients together after compression. Binders for solid pharmaceutical compositions include, for example, acacia, alginic acid, carbomer (e.g. carbopol), carboxymethylcellulose sodium, dextrin, ethyl cellulose, gelatin, guar gum, gum tragacanth, hydrogenated vegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone, pregelatinized starch, sodium alginate, and / or starch, e.g. corn starch.

[0196] The dissolution rate of a compacted solid pharmaceutical composition in the patient's stomach can be increased by the addition of a disintegrant to the composition. Disintegrants include, for example, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, colloidal silicon dioxide, croscarmellose sodium, crospovidone, guar gum, magnesiumaluminum silicate, methyl cellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate, and / or starch, such as corn starch, or potato starch.

[0197] Glidants can be added to improve the flowability of a non-compacted solid composition and to improve the accuracy of dosing. Excipients that can function as glidants include, for example, colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc, and / or tribasic calcium phosphate.

[0198] When a dosage form such as a tablet is made by the compaction of a powdered composition, the composition is subjected to pressure from a punch and dye. Some excipients and active ingredients have a tendency to adhere to the surfaces of the punch and dye, which can cause the product to have pitting and other surface irregularities. A lubricant can be added to the composition to reduce adhesion and ease the release of the product from the dye. Lubricants include, for example, magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, and / or zinc stearate.

[0199] Flavoring agents and / or flavor enhancers make the dosage form more palatable to the patient. Common flavoring agents and / or flavor enhancers for pharmaceutical products that can be included in the composition of the present invention include, for example, maltol, peppermint, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, oil of Wintergreen and / or tartaric acid.

[0200] The pharmaceutical composition can also be dyed using any pharmaceutically acceptable colorant to improve their appearance and / or facilitate patient identification of the product and unit dosage level.

[0201] In liquid pharmaceutical compositions, the crystalline TLR4 agonist of the present invention and any other solid excipients are dissolved or suspended in a liquid carrier such as e.g. water, vegetable oil, alcohol, polyethylene glycol, propylene glycol, or glycerin.

[0202] Liquid pharmaceutical compositions can contain emulsifying agents to disperse an active ingredient or other excipient that is not soluble in the liquid carrier uniformly throughout thecomposition. Emulsifying agents that can be useful in liquid compositions of the present invention include, for example, gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol, and / or cetyl alcohol.

[0203] Liquid pharmaceutical compositions can also contain a viscosity enhancing agent to improve the mouth-feel of the product and / or coat the lining of the gastrointestinal tract. Such agents include, for example, acacia, alginic acid bentonite, carbomer, carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methyl cellulose, ethylcellulose, gelatin guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polyvinyl alcohol, povidone, propylene carbonate, propylene glycol alginate, sodium alginate, sodium starch glycolate, starch tragacanth, and / or xanthan gum.

[0204] Sweetening agents such as sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, lactose, mannitol, and invert sugar can be added to improve the taste.

[0205] Preservatives and chelating agents such as alcohol, sodium benzoate, butylated hydroxyl toluene, butylated hydroxyanisole, and ethylenediamine tetraacetic acid can be added at levels safe for ingestion to improve storage stability.

[0206] A liquid composition can also contain a buffer such as gluconic acid, lactic acid, citric acid, or acetic acid, sodium gluconate, sodium lactate, sodium citrate, or sodium acetate. Selection of excipients and the amounts used can be readily determined by the formulation scientist based upon experience and consideration of standard procedures and reference works in the field.

[0207] The solid compositions of the present invention include powders, granulates, aggregates, and compacted compositions. The dosages include dosages suitable for oral, buccal, rectal, parenteral (including subcutaneous, intramuscular, and intravenous), inhalant, and ophthalmic administration. The most suitable administration in any given case will depend on the nature and severity of the condition being treated. The dosages can be conveniently presented in unit dosage form and prepared by any of the methods well-known in the pharmaceutical arts.

[0208] Dosage forms may include solid dosage forms like tablets, powders, capsules, suppositories, sachets, troches, and lozenges, as well as liquid syrups, suspensions, and elixirs.

[0209] The dosage form of the composition of the present invention may be a capsule containing thecomposition, preferably a powdered or granulated solid composition of the invention, within either a hard or soft shell. The shell can be made from gelatin and optionally contain a plasticizer such as glycerin and sorbitol, and an opacifying agent or colorant.

[0210] The crystalline TLR4 agonist, as an active ingredient, and excipients can be formulated into compositions and dosage forms according to methods known in the art.

[0211] A composition for tableting or capsule filling can be prepared by wet granulation. In wet granulation, some or all of the active ingredients and excipients in powder form are blended and then further mixed in the presence of a liquid, typically water, that causes the powders to clump into granules. The granulate is screened and / or milled, dried, and then screened and / or milled to the desired particle size. The granulate can then be tableted, or other excipients can be added prior to tableting, such as a glidant and / or a lubricant.

[0212] A tableting composition can be prepared conventionally by dry blending. For example, the blended composition of the actives and excipients can be compacted into a slug or a sheet and then comminuted into compacted granules. The compacted granules can subsequently be compressed into a tablet.

[0213] As an alternative to dry granulation, a blended composition can be compressed directly into a compacted dosage form using direct compression techniques. Direct compression produces a more uniform tablet without granules. Excipients that are particularly well suited for direct compression tableting include microcrystalline cellulose, spray dried lactose, dicalcium phosphate, dihydrate, and colloidal silica. The proper use of these and other excipients in direct compression tableting is known to those in the art with experience and skill in particular formulation challenges of direct compression tableting.

[0214] A capsule filling of the present invention can comprise any of the aforementioned blends and granulates that were described with reference to tableting, but they are not subjected to a final tableting step.

[0215] The TLR4 agonist of the present invention alone or in combination with other active agents can be formulated as pharmaceutical compositions, as described above, and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route ofadministration, e.g., orally or parenterally, e.g. by intravenous, intramuscular, topical or subcutaneous routes.

[0216] Thus, the composition comprising the TLR4 agonist according to the present invention, optionally in combination with another active agent, e.g., another adjuvant or an antigen, may be systemically administered, e.g. orally, in combination with a pharmaceutically acceptable excipient, such as an inert diluent or carrier, as described above. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, as described above, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the composition including the TLR4 agonist of the present invention, optionally in combination with another active compound, may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the TLR4 agonist, optionally in combination with another active compound, may be incorporated into sustained-release preparations and devices.

[0217] Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the TLR4 agonist of the present invention and optionally other active compound(s) in such useful compositions is such that an effective dosage level will be obtained.

[0218] Administration of a compositions of the present invention can also be via parenterally administration, for example, intravenously, intra-arterially, intraperitoneally, intrathecal I y, intraventricularly, intraurethrally, intrasterna I ly, intracranially, intramuscularly, or subcutaneously. Such administration may be as a single bolus injection, multiple injections, or as a short- or long-duration infusion. Implantable devices (e.g., implantable infusion pumps) may also be employed for the periodic parenteral delivery over time of equivalent or varying dosages of the particular formulation. For such parenteral administration, the compounds (the TLR4 agonist of the present invention and optionally another active agent) may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances such as salts, sugars (particularly glucose or mannitol), to make the solution isotonic with blood, buffering agents such as acetic, citric, and / or phosphoric acids and their sodium salts, and preservatives.The composition comprising the TLR4 agonist of the present invention, optionally in combination with another active compound, such as an antigen or (a) further adjuvant(s), may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the TLR4 agonist of the present invention, optionally in combination with another active compound, or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. These preparations may also contain a preservative to prevent the growth of microorganisms.

[0219] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient(s) which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms during storage can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0220] Sterile injectable solutions are prepared by incorporating compound(s) in the required amount in the appropriate solvent, optionally with other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, one method of preparation includes vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously steri le-fi Itered solutions.

[0221] For topical administration, the crystalline form of a TLR4 agonist, optionally in combination with another active compound, such as antigen(s) and adjuvant(s), may be applied in pure form, e.g.,when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0222] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Fragrances and antimicrobial agents can be added to enhance the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver compounds to the skin are known to the art; for example, seejacquetetal. (U. S. Pat. No. 4,608,392), Geria (U. S. Pat. No. 4,992,478), Smith et al. (U. S. Pat. No. 4,559,157) and Wortzman (U. S. Pat. No. 4,820,508).

[0223] In addition, in one embodiment, the invention provides various dosage formulations of the TLR4 agonist according to the present invention, optionally in combination with another active compound, such as a further adjuvant or an antigen, for inhalation delivery. For example, formulations may be designed for aerosol use in devices such as metered-dose inhalers, dry powder inhalers and nebulizers.

[0224] Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Pat. No. 4,938,949. The ability of an adjuvant to act as a TLR agonist may be determined using pharmacological models which are well known to the art, including the procedures disclosed by Lee etal., Proc. Natl. Acad. Sci. USA, 100: 6646 (2003).

[0225] The amount of the TLR4 agonist according to the present invention, optionally in combination with another active compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patientand will be ultimately at the discretion of the attendant physician or clinician. In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as about 3 to 50 mg per kilogram body weight of the recipient per day, for instance in the range of about 6 to 90 mg / kg / day, e.g., in the range of about 15 to 60 mg / kg / day.

[0226] The TLR4 agonist(s) according to the present invention, optionally in combination with another active compound, may be conveniently administered in unit dosage form, containing, for example, about 5 to 1000 mg, about 10 to 750 mg, or about 50 to 500 mg of active ingredient per unit dosage form.

[0227] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. The dose, and perhaps the dose frequency, will also vary according to the age, body weight, condition, and response of the individual patient. In general, the total daily dose range for an active agent for the treated condition, may be from about 50 mg to about 5000 mg, in single or divided doses. This can achieve effective plasma levels of about 500-750 uM. In managing the patient, the therapy could be initiated at a lower dose and increased depending on the patient's global response.

[0228] The pharmaceutical composition of the present invention comprising a crystalline form of a TLR4 agonist of the present invention may comprise at least one further active ingredient. In a particular embodiment, the at least one further active ingredient may e.g. be a further adjuvant, such as e.g. a TLR7 agonist. In another particular embodiment, the at least one further active ingredient may be a (further) anti-cancer drug, such as an immunotherapeutic drug. Examples of immunotherapeutic drugs for the treatment of cancer are, without being limited thereto, checkpoint inhibitors, such as e.g. pembrolizumab and ipilimumab.

[0229] The pharmaceutical composition of the present invention comprising a crystalline form of a TLR4 agonist of the present invention may also comprise an antigen. Examples of antigens are, without being limited thereto, a carbohydrate, an amino acid, a peptide, a protein, a nucleic acid, such e.g. an DNA, or an RNA, in particular an mRNA, a lipid, a body substance, or a cell. An example of a specific antigen is a microbe, such as e.g. a virus, bacteria, or fungi. Specific viruses are RNAviruses, such as e.g. RSV and influenza virus, a product of the RNA virus, or a DNA virus, such as e.g. herpes virus or hepatitis B virus.

[0230] Alternatively or additionally, the pharmaceutical composition of the present invention comprising a crystalline form of a TLR4 agonist of the present invention may also comprise a virostatic agent, such as e.g. ribavirin, an immunesuppressive agent, such as e.g. mizoribine, and mycophenolate mofetil.

[0231] The crystalline form of a TLR4 agonist of the present invention, and optionally further active ingredient(s), as described above, may be formulated in liposomes, as e.g. described in W02020 / 186229 A1, the teaching of which is hereby incorporated by reference.

[0232] The liposomes may comprise e.g. 1,2-dioleoyl-s / 7-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoy!-5 / 7-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-5 / ^glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl- / 7-glycero-3-phospho-(1 '-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-5 / 7-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-s / glycero-3- PE), 1,2-dipalmitoyl-5 / 7-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000 PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-5 / ?-glycero-3-phosphocholine (18:1-12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-5 / 7-glycero-3-phosphocholine (16:0-12:0 NBD PC), and mixtures thereof; 1,2-distearoyl-s / >glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-s / ?-glycero-3-phosphoethanolamine (DOPE), cholesterol, or a mixture thereof. In one embodiment, the liposomes comprise DOPC, cholesterol or combinations thereof.

[0233] The present invention further encompasses a process for preparing a pharmaceutical composition comprising combining the crystalline form of a TLR4 agonist of the present invention, with at least one pharmaceutically acceptable excipient, as described above, and optionally with at least one further active ingredient, as described above.

[0234] The beneficial properties of the crystalline forms of a TLR4 agonist according to the present invention in terms of stability, solubility, and hygroscopicity, as well as their beneficial mechanical properties will have an advantageous effect on the process for preparing a pharmaceutical composition according to the present invention.Use of the crystalline forms of the TLR4 agonist or of the pharmaceutical composition comprising the same

[0235] In still another aspect, the present invention provides a crystalline form of the TLR4 agonist according to the present invention, or a crystalline form of the TLR4 agonist as isolated / obtained by a method according to the present invention, or a crystalline form of the TLR4 agonist as prepared by a method according to the present invention, or of a pharmaceutical composition according to the present invention for use as an adjuvant or anti-cancer drug.

[0236] The crystalline form of the TLR4 agonist or the pharmaceutical composition comprising the same may be used as an adjuvant in a vaccine for preventing or treating an infectious disease. Further, the crystalline form of the TLR4 agonist or pharmaceutical composition of the present invention may be used as an anti-cancer drug in an anti-cancer medicament. Administration is usually in an "effective amount", e.g. in a "prophylactical ly effective amount" or in a "therapeutically effective amount" (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature or severity of what is being treated, in particular the nature and severity of an infectious or cancer disease.

[0237] Examples

[0238] The invention will be further described by the following non-limiting examples.

[0239] Example 1: Manufacturing process of 2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-(5,4-b)indol-2-yl)thio)-N-cyclohexylacetamide (2B182C " Pattern A")

[0240] 2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-(5,4-b)indol-2-yl)thio)-N-cyclohexylacetamide (2B182C " Pattern A") was prepared from ethyl 3-amino-5-bromo-1 H-indole-2-carboxylate (Compound 1) as a starting material by an inventive process as shown Scheme 1 below.Compound 1 as a starting material was prepared as reported by M. Chan etai., Structure-Activity Relationship Studies of Pyrimido[5,4- / ?[lindoles as Selective Toll-Like Receptor 4 Ligands, / . Med. Chem., 2017, 60, 9142-9161, the content of which is incorporated herein by reference.

[0241] Scheme 1:

[0242] PhNCS EtOH, reflux

[0243]

[0244] 1: m.w. 269.1 2: m.w. 283.1 3: m.w. 297.1 4: m.w. 419.5 5: m.w. 373.4 6: m.w. 512.6

[0245] Step 1: Synthesis of ethyi-1 -methyl-3-amino-5-bromo-1 H-indoie-2-carboxyiate (2)

[0246] Sodium hydride 60% dispersion in oil (4.4 g, 0.11 mol) was slowly added to a solution of ethyl 3-amino-5-bromo-1 H-indole-2-carboxylate (1, 28.3 g, 0.1 mol) in DMF (280 mL). The reaction mixture was stirred at room temperature for 5 min, and then methyl iodide (6.3 mL, 0.1 mol) was added and stirring was continued at room temperature for 10 min. After addition of water, the methylated product was extracted with ethyl acetate, dried over MgSO4and evaporated to dryness. The crude material was purified by silica gel column chromatography (hexane: EtOAc = 70:30) to give 24.6 g of compound 2 in 82.8% yield.

[0247] Step 2: Synthesis of ethyl 1-methyl-3-amino-5-(2-furyl)-1H-indole-2-carboxylate (3)

[0248] To a solution of compound 2 (1.68 kg, 5.65 mol) and 2-furanboronic acid pinacol ester (877 g, 4.52 mol) in dimethoxyethane (22 L) sodium carbonate (1.9 kg) and water (13.1 L) were added at 25°C and stirring was continued for 2 hours whilst nitrogen was bubbled into the reaction mixture. [1,1 '-bis(diphenylphosphino)ferrocene] dichloropalladium (II) (59 g, 0.08 mol) were added, the temperature was slowly raised to 75°C over 6 hours, then stirring was continued at this temperature for 3 hours. The reaction mixture was cooled to 25°C and suction filtered washing the cake with ethyl acetate (10 L). The filtrate was concentrated under vacuum, keeping the temperature below 40°C, diluted with ethyl acetate (24.5 L) and washed with water (13 L). The organic layer was treated with SiliaMetS® Dimercaptotriazine (0.9 kg) and stirred at 45°Cfor 12 hours, cooled to 20°C and insoluble material was filtered, washing the cake with AcOEt (6 L). The organic layer was washed twice with water (8.5 L) then was decolorated by CUNO High Flow filtration system, evaporated to dryness under vacuum and crystallized from isopropanol to obtain 0.76 kg (45.3% yield) of 3.

[0249] Step 3: Synthesis of ethyl 1-methyl-5-(2-furyl)-3-(3-phenylthioureido)amino-1H-indole-2-carboxylate (4)

[0250] To a solution of 3 (0.76 kg, 2.56 mol) in acetonitrile (5.8 kg) phenyl isothiocyanate (0.48 kg, 3.55 mol) was added and the reaction mixture was stirred at 50°C for 16 hours, then isopropanol (3.2 L) was added drop-wise over 4 hours at the same temperature. After cooling and stirring at 5°C for 10-12 hours, the precipitate was collected and dried to provide compound 4 (1 kg, 93% yield).

[0251] Step 4: Synthesis of 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5, 4-bj- indol-4(5H)-one (5)

[0252] To a stirred mixture of 4 (1.05 kg, 2.5 mol) in anhydrous EtOH (9 L), sodium ethoxide (200 g, 2.95 mol) was added and stirring was continued at 50°C for 6-8 hours. After cooling to 20°C, 1 M hydrochloric acid (5.2 kg) was added slowly during 4 hours to the stirred mixture. The resulting precipitate was collected by suction filtration, washing the resulting cake with water (12 L). The cake was then dried at 50°C for 22 hours, to provide 0.88 kg (2.36 mol, 94.3% yield) of compound 5.

[0253] Step 5: Synthesis of 2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-(5,4-b)indol-2-yl)thio)-N-cyclohexylacetamide (6)

[0254] Compound 5 (940 g, 2.52 mol) was dissolved in anhydrous dimethylformamide (6 L) under nitrogen athmosphere and 2-chloro-N-cyclohexylacetamide (534 g, 3.02 mol) and triethylamine (0.75 L, 5.4 mol) were added. The reaction mixture was stirred at 20 - 30°C for 2 hours. Isopropanol (3.8 L) was added drop-wise over 2 hours followed by purified water (600 mL) added drop-wise over 7 hours. Stirring was continued at 20-30°C for 6 h. The resulting precipitate was collected by suction filtration and washed several times with isopropanol-water 1:1. After drying the wet cake at 55°C in a vacuum oven, 1.38 kg of crude compound 6 were obtained, which were recrystallized from DMSO-i-PrOH to provide 1.13 kg (87.5% yield) of final compound 6.

[0255] The product obtained by the above process (2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5- dihydro-3H-pyrimido-(5,4-b)indol-2-yl)thio)-N-cyclohexylacetamide; compound 6) was named2B182C " Pattern A". Subsequent investigations showed that it was actually a mixture of two polymorphic forms, referred to as Pattern D and Pattern I.

[0256] 2B182C Pattern A (batch PJ02411 -37-FP-DRY), obtained by the method as described above, was further characterized by X-ray diffraction, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and1H-NMR spectrometry.

[0257] Pattern A is an anhydrate with high crystallinity. XRPD shows an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of about 6.91, 13.14, and 16.95, measured using an X-ray wavelength of 1.5406 A (cf. Figure 16). DSC shows a melting peak at Tonset of 260.7°C with an enthalpy of about 91 J / g (cf. Figure 17). TGA shows about 0.5% weight loss at about 170°C, and about 0.3% weight loss from 170°C to 210°C (cf. Figure 18).1H-NMR (DMSO-06) shows 0.3% MTBE residue by weight (cf. Figure 19). A PLM photograph of 2B182C Pattern A (cf. Figure 20) shows a rod-like and plate-like morphology with a particle size ranging from 10-100 pm.

[0258] 2B182C Pattern A (Chemical Formula: C29H28N4O3S; Molecular Weight: 512.63) was used as a starting material in the polymorph screening experiments.

[0259] Example 2: Polymorph Screening Experiments

[0260] Polymorphic transformations of " Pattern A" were investigated under the following conditions:

[0261] 1.1 Determination of approximate solubility at 25°C and at 50°C

[0262] About 5 mg of 2B182C Pattern A were weighed in a 2 mL glass vial. 20 pL aliquots of each solvent were added to dissolve the drug substance at 25 or 50°C. Vortex stirring and sonication were applied to assist dissolution. Maximum volume of each solvent added was 1mL. Approximate solubility was determined by visual observation.

[0263] 1.2 Equilibration with solvents at 25°C

[0264] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were equilibrated in 0.4-2.0 mL of solvents at 25°C for 2 weeks and 3 weeks with a stirring bar on a magnetic stirring plate at a rate of 300-400 rpm. Obtained suspensions were filtered through a 0.45 pm nylon membrane filter by centrifugation at 14,000 rpm. Solid parts (wet cakes) were investigated byXRPD. For samples with different XRPD patterns, additional analyses including HPLC, DSC, TGA, and ’H-NMR were performed.

[0265] 1.3 Equilibration with solvents at 50°C

[0266] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were equilibrated in 0.3-2.5 ml_ of solvents at 50°C for 1 week and 2 weeks with a stirring bar on a magnetic stirring plate at a rate of 300-400 rpm. Obtained suspensions were filtered through a 0.45 pm nylon membrane filter by centrifugation at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD.

[0267] 1.4 Equilibration under a temperature cycle

[0268] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were equilibrated in 0.4-2.0 ml of solvents under a temperature cycle between 5°C to 50°C at a heating / cooling rate of 0.2°C / min for 10 cycles. The equilibration was executed with a stirring bar on a magnetic stirring plate at a rate of 300-400 rpm. Obtained suspensions were filtered through a 0.45 pm nylon membrane filter by centrifugation at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD. For samples with different XRPD patterns, additional analyses including HPLC, DSC, TGA,1H-NMR, KF and PLM were performed.

[0269] 1.5 Crystallization at room temperature by slow evaporation

[0270] Based on approximate solubility results, about 30 mg of 2B182C Pattern A were dissolved in 0.8-20.0 mL of solvents. Clear solution was obtained by filtration through a 0.45 pm syringe nylon membrane filter. The clear solutions were slowly evaporated in ambient condition (about 21-23°C, 30-50% RH). Solid residues were investigated by XRPD and PLM. For samples with different XRPD patterns, additional analysis including HPLC, DSC, TGA, ’H-NMR and PLM was performed.

[0271] 1.6 Crystallization at room temperature by fast evaporation

[0272] Based on approximate solubility results, about 30 mg of 2B182C Pattern A were dissolved in 0.8-20 mL of solvents. Clear solution was obtained by filtration through a 0.45 pm syringe membrane filter. The clear solutions were fast evaporated at room temperature (about 21 -23°C) under a dry nitrogen flow. Solid residues were investigated by XRPD and PLM. For samples with different XRPD pattern, additional analysis including HPLC, DSC, TGA, and ’H-NMR was performed.1.7 Crystallization from hot saturated solutions by slow cooling

[0273] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were dissolved in the minimal amount of selected solvents at 50°C. Clear solution was obtained by filtration through a 0.45 pm syringe membrane filter. The clear solutions were cooled to 5°C at 0.1 °C / min. Samples without precipitates at 5°C were further cooled to -20°C. Precipitates were collected by centrifugation filtration through a 0.45pm nylon membrane filter at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD. For sample with different XRPD pattern, additional analysis including DSC, TGA, and ’H-NMR was performed.

[0274] 1.8 Crystallization from hot saturated solutions by fast cooling

[0275] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were dissolved in the minimal amount of selected solvents at 50°C. Clear solution was obtained by filtration through a 0.45 pm syringe membrane filter. The clear solutions were put into a 0°C ice bath and agitated. Sample without precipitates at 0°C was equilibrated at 5°C for 3 days. Precipitates were collected by centrifugation filtration through a 0.45 pm nylon membrane filter at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD.

[0276] 1.9 Crystallization by addition of anti-solvent

[0277] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were dissolved in the minimal amount of selected good solvents at ambient temperature (about 23°C). Clear solution was obtained by filtration through a 0.45 pm syringe membrane filter. 4-8 folds of anti-solvent were added into the clear solutions slowly. Precipitates were collected by centrifugation filtration through a 0.45 pm nylon membrane filter at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD. For sample with different XRPD pattern, additional analysis including DSC, TGA, and1H-NMR was performed.

[0278] 1.10 Crystallization by reverse addition of anti-solvent

[0279] Based on approximate solubility results, about 50 mg of 2B182C Pattern A) were dissolved in the minimal amount of selected good solvents at ambient temperature (about 23°C). Clear solution was obtained by filtration through a 0.45 pm syringe membrane filter. The clear solutions were added into 4-8 folds of anti-solvent quickly. Precipitates were collected by centrifugation filtration through a 0.45 pm nylon membrane filter at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD.1.11 Crystallization by vapour diffusion

[0280] Based on approximate solubility results, about 50 mg of 2B182C Pattern A were dissolved in the minimal amount of selected solvents at ambient temperature (about 21-23 °C). Clear solution was obtained by filtration through a 0.45 pm syringe membrane filter. The clear solutions were transferred into 4mL glass vials without lids. Then these 4mL lid less vials were placed to 40 mL glass vials. To the 40 mL vials were added anti-solvents. Then these 40 mL vials were capped tightly and placed at ambient temperature for up to 12 days. Precipitates were collected by centrifugation filtration through a 0.45 pm nylon membrane filter at 14,000 rpm. Solid parts (wet cakes) were investigated by XRPD.

[0281] 1.12 Crystallization by heat-cool DSC

[0282] Polymorphic behaviour of 2B182C Pattern A was investigated by two different heat-cool DSC cycles.

[0283] Results

[0284] Using the methods 1.1 to 1.12 described above, in total 11 crystalline forms were identified as either polymorphs or pseudo-polymorphs of Pattern A, including 8 anhydrates, named as Pattern A, C, D, E, F, H, I and L, one hydrate, named as pattern B, one hetero-solvate, named as pattern G, and one intermediate form pattern K. Besides, a crystalline degradation product, pattern J and a degraded amorphous form were obtained. A XRPD overlay of the identified polymorphs and pseudopolymorphs is shown in Figure 21. A thorough analysis of their properties, including purity, stability, pharmaceutical suitability and ease of chemical preparation is reported in the following studies.

[0285] The generation conditions of polymorphs and pseudopolymorphs, and the solvents used in the screening experiments, respectively, are summarized in Table 1 below.Table 1: Generation conditions of polymorphs and pseudopolymorphs

[0286] Polymorphs or Screening experiments

[0287] pseudo-polymorphs

[0288] Pattern B, hydrate From MeOH, toluene, MeOH / water, ACN / water and THF / MeOH by equilibration, slow evaporation, anti-solvent addition and reverse anti-solvent addition experiments

[0289] Pattern C, anhydrate From EtOH, IPA, MTBE, THF / water by equilibration experiments, from DCM, THF / EtOH, MeOH, ACN, MEK, 1,4-dioxane, DCM / EtOH, DCM / acetone, THF / MTBE, THF / heptane, DCM / MTBE and DMAc / EA by slow evaporation, fast evaporation, fast cooling, anti-solvent addition, reverse anti-solvent addition and vapor diffusion experiments

[0290] Pattern D, anhydrate From acetone, EA, DCM by equilibration, slow cooling and fast cooling experiments

[0291] Pattern E, anhydrate From ACN, THF, THF / water (1:1, v:v), and DMF / IPAc (1:1, v:v) by equilibration at25°C (cf. Ex. 1.2);

[0292] from EtOH, IPA, acetone, ACN, THF, DMSO and DMF / IPAc (1:1, v:v) by equilibration at 50°C (cf. Ex. 1.3);

[0293] from ACN, THF, THF / water (1:1, v:v), and DMF / IPAc (1:1, v:v) by equilibration under a temperature cycle (5°C-50°C) (cf. Ex. 1.4); from MEK, THF, DMSO and, DMAc / ACN (1:1, v:v) by crystallization from hot saturated solutions by slow cooling (cf. Ex.

[0294] 1.5);

[0295] from DMAc / ACN (1:1, v:v) by crystallization from hot saturated solutions by fast cooling (cf. Ex. 1.6);

[0296] from DMSO / ACN by crystallization by anti-solvent addition (cf. Ex.

[0297] 1.9) and reverse anti-solvent addition (cf. Ex. 1.10);

[0298] from DMF / MEK by crystallization by vapor diffusion experiments (cf. Ex. 1.11)

[0299] Pattern F, anhydrate From 1,4-dioxane by equilibration at 25°C or 50°C

[0300] Pattern G, heteroFrom DMSO by equilibration experiment

[0301] solvate

[0302] Pattern H, anhydrate From THF, DCM / EA, THF and 1,4-dioxane by slow evaporation, slow cooling and fast cooling experiments

[0303] Pattern I, anhydrate From THF / water and ACN / water by equilibration, from THF,

[0304] acetone, THF / water, 1,4-dioxane / water and DMF / water by fast evaporation, anti-solvent addition and reverse anti-solvent addition experiments

[0305] Pattern J, degradation From acetone by slow evaporation experiment

[0306] product

[0307] Pattern K, From 1,4-dioxane by equilibration (wet cake) experiment intermediate form

[0308]

[0309] Polymorphs or Screening experiments

[0310] pseudo-polymorphs

[0311] Patern L, an hydrate From ACN and ACN / water by competitive equilibration at 25°C;

[0312] from acetone, THF, DMF / IPAc, THF / water and ACN by competitive equilibration at 50°C;

[0313] from THF / water (a.w.=0.3 and a.w.=0.6) by water activity experiments

[0314] Amorphous form, From DCM by fast evaporation experiment

[0315] degradation

[0316]

[0317] ACN=acetonitrile; DCM=dichloromethane; DMAc=dimethylacetamide;

[0318] DMF=dimethylformamide; DMSO=dimethylsulfoxide; EA=ethyl acetate; EtOH=ethanol;

[0319] IPA=isopropanol; IPAc= isopropyl acetate; MEK=methyl ethyl ketone; MeOH= methanol;

[0320] MTBE=methyl-te f-butylether; THF=tetrahydrofuran

[0321] Example 3: Interrelationship investigation of polymorph paterns - Competitive equilibration experiments

[0322] To determine relative stability of various polymorphic forms, competitive equilibration experiments and water activity experiments were conducted as described below.

[0323] 2.1 Competitive equilibration experiments - 1

[0324] To determine relative stability of 2B182C Pattern C, Pattern D, Pattern E, Pattern F, and Pattern H, competitive equilibration experiments were conducted in different solvent systems as indicated in Table 2 below.

[0325] About 10 mg of Pattern C (sample ID FR03350-3-SU2-TC-EtOH-300mg), 10 mg of Pattern D (sample ID FR03350-3-SU3-TC-acetone-300 mg), 10 mg of Pattern E (sample ID FR03350-3-SU4-TC-ACN-300 mg), 10 mg of Pattern F (FR03350-3-SU5-TC-dioxane-300mg), 2 mg of Pattern H (FRO335O-2-SE2-THF) were added to 0.9-1.3 mL of saturated solutions of selected solvents. Obtained suspensions were stirred at 25°C and 50°C. Solid parts (wet cakes) were isolated by centrifugation filtration and investigated by XRPD.

[0326] After equilibration for 26 days, about 2 mg of Pattern L (sample ID FR03350-4-25C-CE8-ACN-water-25d) were added into CE2 (25°C), CE7 (25°C), CE2-CE4 (50°C), and CE7-CE8 (50°C). Obtained suspensions were stirred at 25°C and 50°C. Solid parts (wet cakes) were isolated by centrifugation filtration and investigated by XRPD.After equilibration for 35 days, about 2 mg of Pattern L (sample ID FR03350-4-50C-CE4-THF-35d) were added into CE1 (25°C and 50°C) and CE6 (25°C and 50°C) and CE4 (25°C). Obtained suspensions were stirred at 25°C and 50°C. Solid parts (wet cakes) were isolated by centrifugation filtration and investigated by XRPD. The results are shown in Table 2 below.

[0327] Table 2: Competitive equilibration experiments - 1

[0328] Initial polymorphs: Pattern C, D, E, F, H

[0329] Exp. Solvents XRPD

[0330] ID

[0331] 25°C 50°C

[0332] 5 9 15 25 41 49 5 9 15 25 41 49 days days days days days days days days days days days day s CE1 EtOH E + E + E + E + E + / / E + E + E + E + E + / /

[0333] F F F F F F F F F F CE2 Acetone E + E + E + F F + F + E + E + E + E E / /

[0334] F F F L L F F F CE3 CAN E + E + E + L / / / / E + E + E + E L / /

[0335] F F F F F F CE4 THF E + E + E + E + F + / / E + E + E + E L / /

[0336] F F F F L F F F CE5 1,4- F F / / / / / / / / F F / / / / / / / / dioxane

[0337] CE6 DMF / IPAc E + E + E + E + F + F + E + E + E + E + E + / / (1:1, v:v) F F F F L L F F F F F CE7 THF / water E + E + E + E E / / E + E + E / / E / / (1:1, v:v) F F F F F

[0338] CE8 ACN / water E + E + E + L / / / / E + E / / / / L(m L (1:1, v:v), F F F F ajor) a.w.=0.90* + E

[0339]

[0340] / / : Not carried out.

[0341] As shown in Table 2 above, polymorphs Pattern E, F and L are the most stable forms in several solvents, whereas polymorphs pattern C, D, and H, initially present in the equilibration reaction, turned out to be less stable.2.2 Competitive equilibration experiments - 2

[0342] About 8mg of Pattern E (sample ID FRO335O-3-SU1O-TC-ACN-3OO mg), 8 mg of Pattern F (FRO335O-3-SU11 -TC-dioxane-300 mg), and 8 mg of 2B182C Pattern L (sample ID FR03350-4-SU12-50C-EQ-ACN) were added to 0.5-0.7 mL of saturated solutions of selected solvents. Obtained suspensions were stirred at 25°C and 50°C for 4 days, 9 days, 17 days and 23 days, respectively. Solid parts (wet cakes) were isolated by centrifugation filtration and investigated by XRPD. Results are shown in Table 3 below.

[0343] Table 3: Competitive equilibration experiments - 2

[0344] Initial polymorphs: Pattern E, F and L

[0345] Exp. Solvents XRPD

[0346] ID

[0347] 25°C 50°C

[0348] 4 days 9 days 17 days 23 days 4 days 9 days 17 days CE9 EtOH E + F + L E + F + L E + F + L E + F + L E + F + L L + F L + trace of F CE10 Acetone E + F + L L + F L + F L + F L + F L + trace L of F

[0349] CE11 THF L + F L + F L + F L + F L / / / / CE12 DMF / IPAc L + F L + F L + F L + F L / / / / (1:1, v:v)

[0350] CE13 THF / water L + E + F L + F L + F L + F L + trace L + trace L (1:1, v:v) of F of F

[0351] CE14 ACN L + E + F L + F L + F L + F L + F L + trace L of F

[0352]

[0353] / / : Not carried out.

[0354] As shown in Table 3 above, polymorphs Pattern E, F and L are stable in several solvent systems, and polymorph Pattern L turned out to be the most stable form, especially at 50°C.Example 4: Interrelationship investigation of polymorph paterns - Water activity experiments

[0355] 3.1 Water activity experiments - 1

[0356] To determine critical water activity between 2B182C Pattern B, Pattern E and Pattern F, water activity experiments were conducted at 25°C in different aceton itrile / water (1:1, v:v) systems.

[0357] About 10mg of Pattern B (sample ID FRO335O-2-TC10-toluene and FR03350-2-TC15-MeOH-water), 10 mg of Pattern E (sample ID FR03350-2-TC9-THF and FR03350-2-TC16-THF-water) and 8 mg of Pattern F (sample ID FR03350-3-SU5-TC-dioxane-300mg) were added to 1.5 mL saturated solutions of the ACN / water system. Obtained suspensions were stirred at 25°C for 3 days, 16 days and 26 days, respectively. Solid parts (wet cakes) were isolated by centrifugation filtration and investigated by XRPD. Results are shown in Table 4 below.

[0358] Table 4: Water activity experiments - 1

[0359] Initial polymorphs: Patern B, E and F

[0360] Exp. Solvents XRPD at 25°C after 3 XRPD at 25°C after XRPD at 25°C after ID days 16 days 26 days

[0361] AW1 ACN (a.w.=0)* Pattern E + F Patern E + F Patern E + F AW2 ACN / water Pattern E + F Patern E + F Pattern E + F(major) (a.w.=0.2)*

[0362] AW3 ACN / water Pattern E + F Patern E + F Patern F + 1 peak (a.w.=0.4)*

[0363] AW4 ACN / water Pattern E + F Pattern E + F Pattern F + L (a.w.=0.6)*

[0364] AW5 ACN / water Pattern B + E + F Patern F + 1 peak Pattern F + L (a.w.=0.8)*

[0365] AW6 Water (a.w.=1)* Pattern B + E + F Patern B Pattern B

[0366]

[0367] Note: water activity is calculated by UNIFAC method.

[0368] As can be taken from Table 4 above, patterns E, F, and L are stable at 25°C in all ACN / water systems tested, while the hydrate patern B was only stable in a water system (a.w.=1).3-1 Water activity experiments - 2

[0369] The water activity experiments were conducted at 25°C and 50°C in several THF / water systems to determine critical water activity between 2B182C Pattern B and Pattern L.

[0370] About 8 mg of Pattern B (sample ID FRO335O-2-TC10-toluene and FR03350-2-TC15-MeOH-water), and 8 mg of Pattern L (sample ID FRO335O-4-SU12-25C-EQ-ACN) were added to 0.5-0.8 mL saturated solutions of the THF / water system. Obtained suspensions were stirred at 25°C for 4 days and at 50°C for 1 day, respectively. Solid parts (wet cakes) were isolated by centrifugation filtration and investigated by XRPD. Results are shown in Table 5 below.

[0371] Table 5: Water activity experiments - 2

[0372] Initial polymorphs: Pattern B and Pattern L

[0373] Exp. Solvents XRPD at 25°C after 4 days XRPD at 50°C after 1 day ID AW7 THF / water Pattern L Pattern L

[0374] (a.w.=0.3)*

[0375] AW8 THF / water Pattern L Pattern L

[0376] (a.w.=0.6)*

[0377]

[0378] Note: water activity is calculated by UNIFAC method.

[0379] As can be taken from Table 4 above, Pattern L is stable at 25°C and 50°C in the THF / water systems tested, while the hydrate pattern B is not.

[0380] Example 5: Evaluation of physicochemical properties

[0381] As the results from the above experiments showed that patterns E, F, and L are the more stable forms compared to patterns C, D, and H, respectively, their bulk stability, hygroscopicity and mechanical properties were evaluated.

[0382] 4.1 Bulk stability

[0383] 2B182C Pattern E (sample ID FR03350-3-SU4-TC-ACN-300mg), Pattern F (sample ID FR03350-3-SU5-TC-dioxane-300mg) and Pattern L (sample ID FR03350-3-SU14-EQ-THF-200mg) wereplaced at 25°C / 92% RH in an open container, at 40°C / 75% RH in an open container and at 60°C in a closed container for 12 days. Samples after the stress were characterized by XRPD and HPLC and inspected for color change. Results are summarized in Table 6 below.

[0384] Table 6: Bulk stability

[0385] Polymorph Pattern E Pattern F Pattern L

[0386] Sample ID FR03350-3-SU4-TC- FR03350-3-SU5-TC- FR03350-3-SU14-EQ- ACN-300mg dioxane-300mg THF-200mg

[0387] Exp. Initial purity 99.1% 98.6% 99.8%

[0388] ID

[0389] Initial color Off-white Off-white Off-white

[0390] Purity Color Purity Color Purity Color BS1 Solid state, 25°C / 92%RH, open container, 12 days

[0391] Bulk (HPLC) 98.9% A 98.7% A 99.8% A

[0392] Bulk (XRPD) No form change No form change No form change

[0393] BS2 Solid state, 40°C / 75%RH, open container, 12 days

[0394] Bulk (HPLC) 98.9% A 98.7% A 99.9% A

[0395] Bulk (XRPD) No form change No form change No form change

[0396] BS3 Solid state, 60°C, tight container, 12 days

[0397] Bulk (HPLC) 99.0% A 98.6% A 99.8% A

[0398] Bulk (XRPD) No form change No form change No form change

[0399]

[0400] Explanation A: no change of color; B: slight discoloration

[0401] C: medium discoloration; D: strong discoloration

[0402] As shown in Table 6, polymorph patterns E, F and L showed no change of purity, form and color under the different condition tested, are thus have excellent bulk stability.4.2 Hygroscopicity

[0403] Water sorption and desorption behavior of 2B182C Pattern B (sample ID FR03350-3-SU1 -TC-MeOH-300mg) were investigated by DVS at 25°C with a cycle of 40-95-0-95-40%RH. Water sorption and desorption behavior of Pattern E (sample ID FRO335O-3-SU4-TC-ACN-3OOmg), Pattern F (sample ID FRO335O-3-SU11 -TC-dioxane-300mg) and Pattern L (sample ID FR03350- 3-SU13-EQ-THF-200mg) were investigated by DVS at 25°C with a cycle of 40-0-95-0-40%RH. XRPD were measured after the DVS test to determine form change. The results are presented in Table 7 (for pattern B), Table 8 (for Pattern E), Table 9 (for Pattern F) and Table 10 (for Pattern L).

[0404] Table 7: Water sorption and desorption experiments of Pattern B

[0405] Method 40-95-0-95-40%RH, step: 10%RH, equilibration time 240min per step, 25°C Sample ID FRO335O-3-SU1 -TC-MeOH-300mg

[0406] Relative 1stsorp. Weight % 1stdesorp. Weight 2ndsorp. Weight 2nddesorp. Weight humidity at change % change % change % change 25°C

[0407] 0% N / A 0.1 0.1 N / A

[0408] 10% N / A 0.6 0.6 N / A

[0409] 20% N / A 3.9 3.9 N / A

[0410] 30% N / A 4.4 4.4 N / A

[0411] 40% 4.7 4.8 4.8 4.8

[0412] 50% 4.9 5.0 5.0 5.0

[0413] 60% 5.1 5.2 5.2 5.2

[0414] 70% 5.3 5.4 5.4 5.4

[0415] 80% 5.4 5.5 5.5 5.5

[0416] 90% 5.6 5.6 5.6 5.6

[0417] 95% 5.7 5.7 5.7 5.7

[0418]

[0419] N / A: Not applicable.Table 8: Water sorption and desorption experiments of Pattern E

[0420] Method 40-0-95-0-40%RH, dm / dt: 0.002% / min, step: 10%RH, minimum equilibration time 60 min / step, maximum equilibration time 360 min / step, 25°C Sample ID FR03350-3-SU4-TC-ACN-300mg

[0421] Relative 1stdesorp. Weight 1stsorp. Weight % 2nddesorp. 2ndsorp. Weight humidity at % change change Weight % change % change 25°C

[0422] 0% 0.00 0.00 0.05 0.05

[0423] 10% 0.01 0.01 0.06 0.06

[0424] 20% 0.02 0.02 0.07 0.07

[0425] 30% 0.03 0.03 0.08 0.07

[0426] 40% 0.04 0.04 0.09 0.08

[0427] 50% N / A 0.05 0.11 N / A

[0428] 60% N / A 0.07 0.14 N / A

[0429] 70% N / A 0.09 0.12 N / A

[0430] 80% N / A 0.11 0.13 N / A

[0431] 90% N / A 0.13 0.14 N / A

[0432] 95% N / A 0.16 0.16 N / A

[0433]

[0434] N / A: Not applicable.Table 9: Water sorption and desorption experiments of Pattern F

[0435] Method 40-0-95-0-40%RH, dm / dt: 0.002% / min, step: 10%RH, minimum equilibration time 60 min / step, maximum equilibration time 360 min / step, 25°C Sample ID FR03350-3-SU11-TC-dioxane-300mg

[0436] Relative 1stdesorp. Weight 1stsorp. Weight % 2nddesorp. 2ndsorp. Weight humidity at % change change Weight % change % change 25°C

[0437] 0% 0.01 0.01 0.00 0.00

[0438] 10% 0.03 0.02 0.01 0.01

[0439] 20% 0.04 0.03 0.03 0.02

[0440] 30% 0.06 0.04 0.03 0.03

[0441] 40% 0.07 0.04 0.05 0.04

[0442] 50% N / A 0.05 0.06 N / A

[0443] 60% N / A 0.06 0.09 N / A

[0444] 70% N / A 0.07 0.13 N / A

[0445] 80% N / A 0.09 0.15 N / A

[0446] 90% N / A 0.14 0.18 N / A

[0447] 95% N / A 0.20 0.20 N / A

[0448]

[0449] N / A: Not applicable.Table 10: Water sorption and desorption experiments of Pattern L

[0450] Method 40-0-95-0-40%RH, dm / dt: 0.002% / min, step: 10%RH, minimum equilibration time 60 min / step, maximum equilibration time 360 min / step, 25°C Sample ID FR03350-3-SU13-EQ-THF-200mg

[0451] Relative 1stdesorp. Weight 1stsorp. Weight % 2nddesorp. 2ndsorp. Weight humidity at % change change Weight % change % change 25°C

[0452] 0% 0.03 0.03 0.00 0.00

[0453] 10% 0.04 0.04 0.01 0.02

[0454] 20% 0.05 0.05 0.03 0.03

[0455] 30% 0.07 0.06 0.04 0.04

[0456] 40% 0.08 0.07 0.05 0.05

[0457] 50% N / A 0.08 0.06 N / A

[0458] 60% N / A 0.09 0.08 N / A

[0459] 70% N / A 0.12 0.12 N / A

[0460] 80% N / A 0.15 0.18 N / A

[0461] 90% N / A 0.20 0.21 N / A

[0462] 95% N / A 0.25 0.25 N / A

[0463]

[0464] N / A: Not applicable

[0465] The results presented in Tables 7-10 show that paterns E, F and L are non-hygroscopic or only slightly hygroscopic.

[0466] In particular, 2B182C Pattern E (sample ID FR03350-3-SU4-TC-ACN-300mg) absorbed about 0.16% water from 0% to 95%RH at 25°C and is therefore classified as being non-hygroscopic.

[0467] 2B182C Patern F (sample ID FR03350-3-SU11-TC-dioxane-300mg) absorbed about 0.19% water from 0% to 95%RH at 25°C and is therefore classified as being non-hygroscopic.

[0468] 2B182C Patern L (sample ID FR03350-3-SU13-EQ-THF-200mg) absorbed about 0.2% water from 0% to 95%RH at 25°C and is therefore classified as being was slightly hygroscopic.On the other hand, 2B182C Pattern B (sample ID FR03350-3-SU1-TC-MeOH-300mg) absorbed about 1.0% water from 40% to 95%RH at 25°C and therefore is clearly more hygroscopic compared to patterns E, F, and L.

[0469] No pattern change was observed after the DVS test, that is, after the DVS test, patterns of obtained samples were still the same (verified by XRPD; results not shown).

[0470] 4.3 Solubility

[0471] 5 mg / 10 mg of 2B182C Pattern E (sample ID FR03350-3-SU10-TC-ACN-300mg), Pattern F (sample ID FR03350-3-SU11 -TC-dioxane-300mg) or Pattern L (sample ID FR03350-3-SU13-EQ-THF-200mg) were weighed into a 2 mL glass vial, respectively. 0.5 mL / 0.2 mL of solubility medium was added. The target concentration was 50 mg / mL in DCM and 10 mg / mL in other solvents. Obtained suspensions were stirred at 25°C or 50°C at 400 rpm for 24 hours and then centrifuged at 14,000 rpm for 5min. Supernatants were analyzed by HPLC for solubility. Residual solids (wet cakes) were characterized by XRPD to determine physical form. Results are shown in Table 11 below:Table 11: Solubility experiments for Pattern E, F and L

[0472] Solubility at 25°C and 50°C, target concentration: 50 mg / mL in DCM and 10 mg / mL in other solvents, equilibration for 24 hours, LOQ: 0.25 pg / mL

[0473] Physical Form

[0474] Pattern E, anhydrate Pattern F, Pattern L, anhydrate anhydrate

[0475] Sampl FRO335O-3-SU10-TC-ACN- FRO335O-3-SU5- FRO335O-3-SU13-EQ-THF- e ID 300mg TC-dioxane- 200mg

[0476] 300mg

[0477] Temp. 25°C 50°C 25°C 25°C 50°C Solven SoluXRPD SoluXRPD SoluXRPD SoluXRPD SoluXRPD t bility (patbility (patbility (patbility (patbility (pat(mg / tern) (mg / tern) (mg / m tern) (mg / tern) (mg / tern) mL) mL) L) mL) mL) ACN 0.98 E 1.63 E 0.75 F 0.55 L 1.45 L THF >10 / / >10 / / 5.90 F 5.21 L >10 / / EtOH 0.52 E 0.87 E 0.44 F 0.34 L 0.89 L MeO 0.57 E 0.80 E + B / / / / 0.40 L 1.02 L + B H DCM >50 / / >50 / / / / / / 31.2 L >50 / /

[0478] 6

[0479]

[0480] ACN=acetonitrile; DCM=dichloromethane; EtOH=ethanol; MeOH=methanol;

[0481] THF=tetrahydrofuran;

[0482] / / : Not carried out

[0483] As can be taken from Table 11 above, Pattern L showed lower solubility in some organic solvents than Pattern F and Pattern E at 25°C, which indicates that Pattern L should be more stable than Pattern F and Pattern E at 25°C.

[0484] 4-4 Mechanical properties

[0485] 4-4.1 Compression simulation experiments

[0486] About 10 mg of 2B182C Pattern L (sample ID FR03350-3-SU14-EQ-THF-200mg) were compressed for 5 minutes under 2MPa, 5MPa and 10MPa with a hydraulic press. Potential formchange and degree of crystallinity were evaluated by XRPD. The results are shown in Table 12 below.

[0487] Table 12: Compression simulation experiments

[0488] Pressure XRPD Comments

[0489] Exp. ID

[0490] CSE1 2MPa Pattern L No obvious change in crystallinity CSE2 5MPa Pattern L No obvious change in crystallinity CSE3 10MPa Pattern L No obvious change in crystallinity

[0491]

[0492] 4-4.2 Dry grinding simulation experiments

[0493] About 20 mg of 2B182C Pattern L (sample ID FR03350-3-SU14-EQ-THF-200mg) were ground manually with a mortar and a pestle for 5min. Potential form change and degree of crystallinity were evaluated by XRPD. The results are represented in Table 13 below.

[0494] Table 13: Dry grinding simulation experiments

[0495] Exp. ID Grinding time XRPD Comments

[0496] GSE1 5min Pattern L No obvious change in crystallinity

[0497]

[0498] 4-4.3 Wet granulation simulation experiments

[0499] Water or ethanol were added drop wise to about 20 mgof 2B182C Pattern L (sample ID FR03350-3-SU14-EQ-THF-200mg) until the sample were wetted sufficiently. Wet sample were ground gently with in a mortar and a pestle. Post granulation sample were dried under ambient condition for 10min. Potential form change and degree of crystallinity were evaluated by XRPD. Results are presented in Table 14 below.Table 14: Wet granulation simulation experiments

[0500] Exp. ID Granulation solvents XRPD Comments

[0501] GNSE1 Water Pattern L No obvious change in crystallinity

[0502] GNSE2 Ethanol Pattern L No obvious change in crystallinity

[0503]

[0504] As shown in Tables 12-14 above, Pattern L (sample ID FR03350-3-SU14-EQ-THF-200mg) showed good tolerance to compression, dry grinding and granulation with no form change and no obvious crystallinity decrease.

[0505] Example 6: Preparation and characterization of polymorph Pattern E

[0506] The various polymorphic crystal forms isolated as described above were prepared in higher amounts from the starting material (Pattern A) using the previously isolated polymorphs as seeds.

[0507] Pattern E is an anhydrate. As described in Example 2, Pattern E can be obtained from ACN, THF, THF / water, EtOH, IPA, acetone, DMSO and DMF / IPAc by equilibration, from MEK, THF, DMSO, DMAc / ACN, DMSO / ACN and DMF / MEK by slow cooling, fast cooling, anti-solvent addition, reverse anti-solvent addition and vapor diffusion experiments.

[0508] For the preparation of higher amounts of Pattern E, 300 mg of Pattern A (batch PJ02411-37-FP-DRY) was weighed into a 20 mL glass vial. About 2 mg seeds of Pattern E (sample ID FR03350-2-TC7-ACN) and 2.5 mL of ACN were added into the vial. After stirring the resulting suspension at 25°C for 2 min, about 2 mg seeds of Pattern E (sample ID FR03350-2-TC7-ACN) were added. The sample was equilibrated under a temperature cycle between 5°C and 50°C at a heating / cooling rate of 0.1 °C / min. After stirring for 1 day, the suspension became stickier, and about 1.5 ml of ACN were added. After stirring for 4 hours, the suspension was filtered by centrifugation through a 0.45 pm nylon membrane filter at 4,000 rpm for 10 min. The solids were dried at ambient condition (25°C, 20%RH) for 1 day. 292.4 mg of Pattern E were obtained as an off-white solid in 97.5% yield.Pattern E was characterized by X-ray diffraction, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and1H-NMR spectrometry.

[0509] Pattern E (sample ID FR03350-3-SU4-TC-ACN-300mg) is of high crystallinity. XRPD shows an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 29 of about 6.56, 13.76, and 17.36, measured using an X-ray wavelength of 1.5406 A. Further characteristic peaks are observed at a reflection angle 29 of 6.90°, 9.16°, and 13.12°. An exemplary XRPD pattern is shown in Figure 1.

[0510] DSC shows a melting peak at Tonsetof 246.9°C, combined with a recrystallization peak at Tonsetof 251.4°C, after recrystallization, it converted to Pattern C. Then, it melts at Tonset of 260.7°C with an enthalpy of about 95J / g. A representative DSC thermogram of Pattern E is shown in Figure 2.

[0511] TGA shows about 0.6% weight loss at about 230°C. A corresponding TGA thermogram of Pattern E is shown in Figure 3.

[0512] 'H-NMR (DMSO-o6) shows no detectable residual solvent. A corresponding ’H-NMR spectrum is shown in Figure 4.

[0513] Pattern E converted to Pattern L at 50°C in competitive equilibration experiments (cf. Example 3).

[0514] A PLM photograph of 2B182C Pattern E presented in Figure 5 shows an aggregated rod-like morphology with a particle size ranging from 10-70 pm. The physicochemical properties of Pattern E are summarized in Table 15 below.Table 15: Characterization of 2B182C Pattern E

[0515] Sample ID FRO335O-3-SU4-TC-ACN-3OO mg

[0516] Parameter Method Result

[0517] Purity HPLC 99.1%

[0518] X-ray diffraction XRPD, 3-40° (2 theta) High crystallinity, Figure 1

[0519] Melting Tonset ® 246.9°C, combined with recrystallization Thermal events

[0520] DSC, 10°C / min

[0521] and enthalpy Tonset ® 251.4°C; melting Tonset @

[0522] 260.7°C, enthalpy about 95J / g, Figure 2

[0523] Thermogravimetry TGA, 10°C / min 0.6% @ 230°C, Figure 3

[0524] No detectable residual solvent, Residual solvents1H-NMR (DMSO-4)

[0525] Figure 4

[0526]

[0527] Example 7: Preparation and characterization of polymorph Pattern F

[0528] Pattern F is an anhydrate. As described in Example 2, Patern F can be obtained from 1,4-dioxane by equilibration experiment at 25°C and 50°C.

[0529] For the preparation of higher amounts of Pattern F, 300 mg of Patern A (batch PJ02411-37-FP- DRY) was weighed into an 8 mL glass vial. About 2.1 mL of 1,4-dioxane were added into the vial. After stirring the resulting suspension at 25°C for 2min, about 2 mg seeds of Patern F (sample ID FR03350-2-TC11 -dioxane) were added. The sample was equilibrated under a temperature cycle between 5°C and 50°C at a heating / cooling rate of 0.1 °C / min. After stirring for 1 day, an additional amount (2 mg) of Pattern F seeds (sample ID FR03350-2-TC11 -dioxane) was added. After stirring for 4 days, the suspension was filtered by centrifugation through a 0.45pm nylon membrane filter at 4,000 rpm for 10 min. The solids were dried at ambient condition (25°C, 20%RH) for 1 day. 284.1 mg of Patern F was obtained as an off-white solid in 94.7% yield.

[0530] Patern F (sample ID FR03350-3-SU5-TC-dioxane-300mg) is of high crystallinity. XRPD shows an X-ray powder diffraction patern comprising characteristic peaks at a reflection angle 20 of about 6.73, 10.88 and 15.49, measured using an X-ray wavelength of 1.5406 A. Further characteristic peaks are observed at a reflection angle 20 of 7.32°, 9.71°, 19.05, 20.50 and 21.38. An exemplary XRPD patern is shown in Figure 6.DSC shows a recrystallization peak at Tonsetof 195.3°C with an enthalpy of about 24J / g, after recrystallization, it converted to Pattern C. Then, it melts at Tonset of 260.2°C with an enthalpy of about 94J / g. A representative DSC thermogram of Pattern F is shown in Figure 7.

[0531] TGA shows about 0.6% weight loss at about 175°C and about 0.6% weight loss from 175°C to 240°C. A corresponding TGA thermogram of Pattern F is shown in Figure 8.

[0532] ’H-NMR shows 0.5% 1,4-dioxane residue by weight. A corresponding ’H-NMR spectrum is shown in Figure 9.

[0533] Pattern F converted to Pattern L at 50°C in competitive equilibration experiments (cf. Example 3).

[0534] A PLM photograph of 2B182C Pattern F presented in Figure 10 shows an aggregated rod-like morphology with a particle size ranging from 10-40 pm. The physicochemical properties of Pattern E are summarized in Table 16 below.

[0535] Table 16: Characterization of 2B182C Pattern F

[0536] Sample ID FR03350-3-SU5-TC-dioxane-300 mg

[0537] Parameter Method Result

[0538] Purity HPLC 98.6%

[0539] High crystallinity, Pattern X-ray diffraction XRPD, 3-40° (2 theta)

[0540] F, Figure 6 Recrystallization Tonset @ 195.3°C, enthalpy Thermal events and about 24J / g; melting DSC, 10°C / min

[0541] enthalpy Tonset @ 260.2°C, enthalpy about 94J / g, Figure 7

[0542] 0.6% @ 175°C; 0.6% Thermogravi metry TGA, 10°C / min from 175°C to 240°C,

[0543] Figure 8

[0544] 0.5% 1,4-dioxane residue by weight (0.03 Residual solvents ’H-NMR (DMSO-ofc)

[0545] equiv. by molar ratio), Figure 9

[0546]

[0547] Example 8: Preparation and characterization of polymorph Patern L

[0548] Pattern L is an anhydrate. It can be obtained from ACN and ACN / water by competitive equilibration at 25°C (cf. Table 2), from acetone, THE, DMF / IPAc, THF / water and ACN by competitive equilibration at 50°C (cf. Table 3), and from THF / water (a.w.=0.3 and a.w.=0.6) by water activity experiments (cf. Table 5), as described above.

[0549] For the preparation of higher amounts of Pattern L, 200 mg of Patern A (batch PJ02411-37-FP-DRY) was weighed into an 8 mL glass vial. About 1.0 ml of THF was added into the vial. After stirring the resulting suspension at 50°C for 2 min, about 2 mg seeds of Pattern L (sample ID FR03350-4-50C-CE4-THF-35d) were added and the sample was equilibrated at 50°C at a rate of 400 rpm. After stirring for 1 day, an additional amount of 2 mg of Pattern L seeds and 0.7 mL of THF were added and stirring was continued for 4 days. The suspension was filtered by centrifugation through a 0.45 pm nylon membrane filter at 4,000 rpm for 10 min, providing 180.5 mg of Patern L, as an off-white solid, in 90.2% yield.

[0550] Patern L (sample ID FR03350-3-SU14-EQ-THF-200mg) is of medium crystallinity. XRPD shows an X-ray powder diffraction patern comprising characteristic peaks at a reflection angle 29 of about 6.46, 10.73, and 19.40, measured using an X-ray wavelength of 1.5406 A. Further characteristic peaks are observed at a reflection angle 20 of 15.5°, 21.5° and 23.0°. An exemplary XRPD patern is shown in Figure 11.

[0551] DSC shows a recrystallization peak at Tonset of 212.8°C with an enthalpy of about 22J / g, after recrystallization, it converted to Patern C. Then, it melts at Tonsetof 260.4°C with an enthalpy of about 99J / g. A representative DSC thermogram of Patern L is shown in Figure 12.

[0552] TGA shows about 0.8% weight loss at about 180°C. A corresponding TGA thermogram of Patern F is shown in Figure 13.

[0553] ’H-NMR shows no detectable residual solvent. A corresponding1H-NMR spectrum is shown in Figure 14.

[0554] A PLM photograph of 2B182C Pattern L presented in Figure 15 shows an aggregated rod-like morphology with a particle size ranging from 10-40 pm. The physicochemical properties of Pattern L are summarized in Table 17 below.Table 17: Characterization of 2B182C Pattern L

[0555] Sample ID FR03350-3-SU14-EQ-THF-200 mg

[0556] Parameter Method Result

[0557] Purity HPLC 99.8%

[0558] Medium crystallinity, Pattern L, X-ray diffraction XRPD, 3-40° (2 theta)

[0559] Figure 11

[0560] Recrystallization Tonset® Thermal events and 212.8°C, enthalpy about 22 J / g;

[0561] DSC, 10°C / min

[0562] enthalpy melting Tonset® 260.4°C, enthalpy about 99 J / g, Figure 12 Thermogravi metry TGA, 10°C / min 0.8% @ 180°C, Figure 13

[0563] No detectable residual solvent, Residual solvents ’H-NMR (DMSO-cfc)

[0564] Figure 14

[0565]

[0566] Although Pattern L was initially obtained at 25°C, conversion during competitive experiments between Pattern E, Pattern F and Pattern L was not completed. Pattern L showed lower solubility in some organic solvents than Pattern F and Pattern E at 25°C, which indicated that Pattern L should be more stable than Pattern F and Pattern E at 25°C. Moreover, 2B182C Pattern L is the most stable polymorph identified at 50°C. It has good chemical and physical stability and is only slightly hygroscopic. Meanwhile, it shows good tolerance to mechanical force.

[0567] Solubility of both Pattern L and Pattern E are comparable in potential solvents used in liposome encapsulate formulation, and Pattern E shows better solubility than Pattern L in dichloromethane. Therefore, it is feasible to select an alternative polymorphic form rather than the most stable form with consideration of process and formulation developability.

[0568] Example 9: Preparation and characterization of polymorph patterns B, C and D (comparative)

[0569] 8.1 Polymorph pattern B

[0570] Pattern B is a hydrate. It was obtained from MeOH, toluene, MeOH / water, ACN / water and THF / MeOH by equilibration, slow evaporation, anti-solvent addition and reverse anti-solvent addition experiments.For the preparation of higher amounts of pattern B, 300 mg of Pattern A was weighed into a 20 mL glass vial. About 2 mg seeds of Pattern B (sample ID FR03350-2-TC2-MeOH) and 8 mL of MeOH were added into the vial. The resulting suspension was stirred at 25°C for 2 min, then about 2 mg seeds of Pattern B were added. After stirring for 10 min, the suspension became stickier. About 3 mL of MeOH was added (suspension). The sample was equilibrated under a temperature cycle between 5°C and 50°C at a heating / cooling rate of 0.1 °C / min (suspension). After stirring for 1 day, the suspension was centrifuged by centrifugation filtration through a 0.45 pm nylon membrane filter at 4,000 rpm for 10 min. The solids were dried at ambient condition (25°C, 20%RH) for 1 day. About 268.0 mg of Pattern B were obtained as an off-white solid in 86.8% yield.

[0571] Pattern B (sample ID FR03350-2-TC2-MeOH) is of high crystallinity. It contains about 3.8% of water by weight (1.1 equiv. by molar ratio) according to KF result. DSC shows a dehydration peak from about 24°C with an enthalpy of about 61 J / g and a melting peak at Tonsetof 260.3 °C with an enthalpy of about 90J / g. TGA shows about 3.7% weight loss at about 150°C. ’H-NMR shows no detectable residual solvent. After dehydration by heating and cooling to ambient condition, Pattern B absorbed water back to Pattern B. Patten B is a metastable form and converted to different anhydrate forms (Pattern E, Pattern F or Pattern L) in a.w. <0.8 at 25°C in water activity experiments.

[0572] The physicochemical properties of pattern B are summarized in Table 18 below.

[0573] Table 18: Characterization of 2B182C Pattern B

[0574] FRO335O-3-SU1-TC- Sample ID

[0575] MeOH-300 mg

[0576] Parameter Method Result

[0577] High crystallinity, Pattern B, Figure X-ray diffraction XRPD, 3-40° (2 theta)

[0578] 22

[0579] Dehydration from about 21 °C, Thermal events and enthalpy about 38J / g; melting DSC, 10°C / min

[0580] enthalpy Tonset ® 260.5°C, enthalpy about 86J / g

[0581] Thermogravi metry TGA, 10°C / min 1.1% © 150°C

[0582] Residual solvents ’H-NMR (DMSO-ofe) No detectable residual solvent 2.9% water by weight (0.9 equiv. Water content Karl Fischer (coulometric)

[0583] by molar ratio)

[0584] Purity HPLC 99.8%

[0585]

[0586] 8.2 Polymorph patern C

[0587] Patern C is an anhydrate. It was obtained from EtOH, IPA, MTBE, THF / water by equilibration experiments, from DCM, THF / EtOH, MeOH, ACN, MEK, 1,4-dioxane, DCM / EtOH, DCM / acetone, THF / MTBE, THF / heptane, DCM / MTBE and DMAc / EA by slow evaporation, fast evaporation, fast cooling, anti-solvent addition, reverse anti-solvent addition and vapor diffusion experiments.

[0588] For the preparation of higher amounts of patern C, 300 mg of Patern A (batch PJ02411-37-FP- DRY) was weighed into a 20 mL glass vial. About 2 mg seeds of Patern C (sample ID FR03350- 2-TC3-EtOH) and 3 mL of EtOH were added into the vial. After stirring the resulting suspension at 25°C for 2 min, about 2mg seeds of Patern C (sample ID FR03350-2-TC3-EtOH) were added. The suspension was equilibrated under a temperature cycle between 5°C and 50°C at a heating / cooling rate of 0.1 °C / min. After stirring for 6 days, the suspension was filtered by centrifugation through a 0.45 pm nylon membrane filter at 4,000 rpm for 10 min. The solids were dried at ambient condition (25°C, 20%RH) for 1 day. 288.3 mg of Patern C were obtained as an off-white solid in 96.1% yield.

[0589] Pattern C (sample ID FR03350-3-SU2-TC-EtOH-300 mg) is of high crystallinity. DSC shows a melting peak at Tonset of 260.4°C with an enthalpy of about 93J / g. TGA shows about 0.2% weight loss at about 250°C. ’H-NMR shows no detectable residual solvent. Pattern C was the desolvation product of Patern G. Patern C should be a stable form at high temperature due to Patern E, Patern F, Pattern H and Patern L converted to Patern C after heating. But, it is a metastable form and converted to Patern E or Patern F in at 25°C and 50°C. The physicochemical properties of patern C are summarized in Table 19 below.

[0590] Table 19: Characterization of 2B182C Patern C

[0591] FR03350-3-SU2-TC- Sample ID

[0592] EtOH-300 mg

[0593] Parameter Method Result

[0594] High crystallinity, Patern C, X-ray diffraction XRPD, 3-40° (2 theta)

[0595] Figure 23

[0596] Thermal events and Melting Tonset ® 260.8°C,

[0597] DSC, 10°C / min

[0598] enthalpy enthalpy about 96J / g Thermogravi metry TGA, 10°C / min 0.2% @ 23O°C

[0599] Residual solvents1H-NMR (DMSO-ofe) No detectable residual solvent

[0600]

[0601] 8.3 Polymorph patern D

[0602] Patern D is an anhydrate. It was obtained from acetone, EA, DCM by equilibration, slow cooling and fast cooling experiments.

[0603] For the preparation of higher amounts of patern D, 300 mg of Patern A (batch PJ02411-37-FP-DRY) was weighed into a 20 mL glass vial. About 2 mg seeds of Patern D (sample ID FRO335O-2-TC5-acetone) and 2 mL of acetone were added into the via). After stirring the resulting suspension at 25°C for 2 min, about 2 mg seeds of Pattern D (sample ID FR03350-2-TC5-acetone) were added. After stirring for 10 min, the suspension became stickier and about 1.5 mL of acetone were added. The suspension was equilibrated under a temperature cycle between 5°C and 50°C at a heating / cooling rate of 0.1 °C / min. After stirring for 1 day, the suspension was filtered by centrifugation through a 0.45pm nylon membrane filter at 4,000 rpm for 10 min. The solids were dried at ambient condition (25°C, 20%RH) for 1 day. 290.3 mg of Patern D were obtained as an off-white solid in 96.8% yield.

[0604] Patern D (sample ID FR03350-2-TC5-acetone) is of high crystallinity. DSC shows a melting peak at Tonset of 260.1 °C with an enthalpy of about 88J / g. TGA shows about 0.6% weight loss at about 170°C and about 0.2% weight loss from 170°C to 210°C.1H-NMR shows 0.2% acetone residue by weight. It is a metastable form and converted to Patern E or Patern F at 25°C and 50°C in competitive experiments. The physicochemical properties of patern D are summarized in Table 20 below.

[0605] Table 20: Characterization of 2B182C Pattern D

[0606] FRO335O-3-SU3-TC- Sample ID

[0607] acetone-300 mg

[0608] Parameter Method Result

[0609] High crystallinity, Patern D, X-ray diffraction XRPD, 3-40° (2 theta)

[0610] Figure 26

[0611] Melting Tonset ® 260.6°C, Thermal events and enthalpy DSC, 10°C / min

[0612] enthalpy about 92J / g

[0613] 1.3% @ 170°C, 0.6% from 170°C Thermogravi metry TGA, 10°C / min

[0614] to 210°C

[0615] 0.1% acetone residue by weight Residual solvents ’H-NMR (DMSO-ofe)

[0616] (0.01 equiv. by molar ratio)

[0617]

Claims

Claims1. A crystalline form of theTLR4 agonist according to Formula (1):Formula (1),selected from the group consisting of(i) a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E);(ii) a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F); and(iii) a crystalline form of the TLR4 agonist according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

2. The crystalline form of the TLR4 agonist according to claim 1, which is a crystalline form according to (i), and which has an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

3. The crystalline form of the TLR4 agonist according to claim 1 or 2, which is a crystalline form according to (i), and which has a melting peak at Tonsetof 246.9°C ±2°C and a recrystallization peak at Tonset of 251,4°C ± 2°C as measured by differential scanning calorimetry.

4. The crystalline form of the TLR4 agonist according to any one of claims 1 to 3, which is a crystalline form according to (i), and which has a relative change in mass of about 0.6% at 230°C as determined by thermal gravimetric analysis.

5. The crystalline form of the TLR4 agonist according to claim 1, which is a crystalline form according to (ii), and which has an X-ray powder diffraction pattern comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

6. The crystalline form of the TLR4 agonist according to claim 1 or 5, which is a crystalline form according to (ii), and which has a recrystallization peak at Ton5et of 195.3°C + 2°C as measured by differential scanning calorimetry.

7. The crystalline form of the TLR4 agonist according to any one of claims 1, 5 and 6, which is a crystalline form according to (ii), and which has a relative change in mass of about 0.6% at 175°C, and of about 0.6% from 175°C to 240°C as determined by thermal gravimetric analysis.

8. The crystalline form of the TLR4 agonist according to claim 1, which is a crystalline form according to (iii), and which has an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 20 as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A.

9. The crystalline form of the TLR4 agonist according to claim 1 or 8, which is a crystalline form according to (iii), and which has a recrystallization peak at Tonsetof 212.8°C + 2°C as measured by differential scanning calorimetry.

10. The crystalline form of the TLR4 agonist according to any one of claims 1, 8 and 9, which is a crystalline form according to (iii), and which has a relative change in mass of about 0.8% at 180°C as determined by thermal gravimetric analysis.

11. A method of obtaining a crystalline form of a TLR4 agonist as represented by FormulaFormula (1),comprising the step of suspending a starting material comprising a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 20 of 6.9, and 16.9 + 0.2° 20, and more preferably comprising characteristic peaks at a reflection angle 20 of 6.9, 13.1, and 16.9 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern A), in a solvent, and obtaining a crystalline form of a compound according to Formula (1 ), which is different from the crystalline form of the starting material, by precipitation, wherein(i) the solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), dimethylformamide / isopropyl acetate (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide, methyl ethyl ketone, dimethylacetamide / acetonitrile (1:1, v:v), to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E); or(ii) the solvent is selected from 1,4-dioxane to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F).

12. The method according to claim 11, which comprises suspending the starting material in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), and dimethylformamide / isopropyl acetate (1:1, v:v), and equilibrating the suspension at a temperature of about 25°C, preferably over a period of 2-3 weeks, thereby obtaining a crystalline form of a compound accordingto Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 + 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

13. The method according to claim 11, which comprises suspending the starting material in a solvent selected from the group consisting of ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, dimethylsulfoxide, and dimethylformamide / isopropylacetate (1:1, v:v), and equilibrating the suspension at a temperature of about 50°C, preferably over a period of 1 -2 weeks, thereby obtaining a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 26, preferably a crystalline form of a compound according to Formula (1) having an X- ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

14. The method according to claim 11, which comprises suspending the starting material in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), and dimethylformamide / isopropylacetate (1:1, v:v), and equilibrating the suspension under a temperature cycle between about 5°C and about 50°C, preferably at a heating / cooling rate of about 0.2 °C / min, preferably over about 10 cycles, thereby obtaining a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

15. The method according to claim 11, which comprises suspending the starting material in a solvent selected from the group consisting of methyl ethyl ketone, tetrahydrofuran, dimethylsulfoxide, and dimethylacetamide / acetonitrile (1:1, v:v) ata temperature of about 50°C, and slow cooling the solution to a temperature of about 5°C to -20°C, preferably at a rate of about 0.1 °C / min, thereby obtaining a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably acrystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

16. The method according to claim 11, which comprises suspending the starting material in a mixture of dimethylacetamide / acetonitrile (1:1, v:v) (1:1 v / v) at a temperature of about 50°C, and fast cooling the solution to a temperature of about 0°C, thereby obtaining a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 + 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

17. The method according to claim 11, which comprises suspending the starting material in dimethylsulfoxide at ambient temperature and adding acetonitrile as an anti¬ solvent, thereby obtaining a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

18. A crystalline form of a compound according to Formula (1 )Formula (1)having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristicpeaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° + 0.2° 29, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, which is obtained by a method according to any one of claims 11 to 17.

19. The method according to claim 11, which comprises suspending the starting material in 1,4-dioxane and equilibrating the suspension in 1,4-dioxane to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 29, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 + 0.2° 29, as measured by X-ray powder diffraction using an X- ray wavelength of 1.5406 A (Pattern F).

20. The method according to claim 19, which comprises equilibrating the starting material suspended in 1,4-dioxane at a temperature of about 25°C for about two weeks.

21. The method according to claim 19, which comprises equilibrating the starting material suspended in 1,4-dioxane at a temperature of about 50°C for about one week.

22. The method according claim 19, which comprises equilibrating the starting material suspended in 1,4-dioxane under a temperature cycle between about 5°C and about 50°C, preferably at a heating / cooling rate of about 0.2 °C / min, preferably for about 10 cycles.

23. A crystalline form of a compound according to Formula (1)Formula (1 )having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 29, preferably a crystalline form of a compound according toFormula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 + 0.2° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A, which is obtained by a method according to any one of claims 11, 19 to 22.

24. A method of obtaining a crystalline form of the TLR4 agonistrepresented by Formula (1):Formula (1),comprising the step of competitively equilibrating a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 26 (Pattern E), and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 26 (Pattern F), in a solvent selected from acetonitrile, aceton itrile / water (1:1, v:v), tetrahydrofuran, acetone, dimethylformamide / isopropylacetate (1:1, v:v), and tetrahydrofuran / water (1:1, v:v) to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 26, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

25. The method according to claim 24, wherein the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 26 (Pattern E), and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 26 (Pattern F) are competitively equilibrated in a solvent selected from acetonitrile and acetonitrile / water (1:1, v:v) at a temperature of about 25°C to obtain a crystalline formof a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 + 0.2° 20, preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 + 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

26. The method according to claim 24, wherein the crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 20 (Pattern E), and a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 + 0.2° 20 (Pattern F) are competitively equilibrated in a solvent selected from acetonitrile, acetonitrile / water (1:1, v:v), tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), acetone, and dimethylformamide / isopropylacetate (1:1, v.v), at a temperature of about 50°C to obtain a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

27. A crystalline form of a compound according to Formula (1)Formula (1)having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L), which is obtained by a method according to any one of claims 24 to 26.

28. A method of preparing a crystalline form of the TLR4 agonist according to FormulaFormula (1),as defined in any of claims 1 to 10, wherein the method comprises suspending a starting material comprising at least one crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at a reflection angle 26 of 6.9, and 16.9 ± 0.2° 26, and preferably comprising characteristic peaks at a reflection angle 26 of 6.9, 13.1, and 16.9 ± 0.2° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern A), in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v.v), ethanol, isopropanol, acetone, dimethylsulfoxide, dimethylformamide / isopropylacetate (1:1, v:v), and 1,4-dioxane, and equilibrating the suspension in presence of a crystalline form of the TLR4 agonist as defined in any of claims 1 to 10 or obtainable by a method according to any one of claims 11-17, 19-22 and 24-26 as a seed.

29. The method according to claim 28, which comprises suspending the starting material in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran, tetrahydrofuran / water (1:1, v:v), ethanol, isopropanol, acetone, dimethylsulfoxide and dimethylformamide / isopropylacetate (1:1, v:v), in the presence of a small amount of a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 26 as a seed, thereby obtaining a larger amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.6, 13.8, and 17.4 ± 0.2° 26, preferably a crystalline form of a compound according to Formula (1) having X-ray powder diffraction pattern comprising characteristic peaks at 6.6°, 6.9°, 9.2°, 13.1°, 13.8°, and 17.4° + 0.2° 26, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern E).

30. The method according to claim 29, wherein the solvent is acetonitrile.

31. The method according to claim 29 or 30, wherein the suspension is equilibrated under a temperature cycle between about 5°C to about 50°C, preferably at a heating / cooling rate of about 0.1 °C / min.

32. The method according to claim 28, which comprises suspending the starting material in 1,4-dioxane, in the presence of a small amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, thereby obtaining a larger amount of a crystalline form of a compound according to Formula (1) having an X- ray powder diffraction pattern comprising characteristic peaks at 6.7, 10.9, and 15.5 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.7°, 7.3°, 9.7°, 10.9°, 15.5°, 19.1, 20.5, and 21.4 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern F).

33. The method according to claim 32, wherein the suspension is equilibrated under a temperature cycle between about 5°C and about 50°C preferably at a heating / cooling rate of about 0.1 °C / min.

34. The method according to claim 28, which comprises suspending the starting material in tetrahydrofuran, and equilibrating the suspension in the presence of a small amount of a crystalline form of a compound according to Formula (1 ) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, thereby obtaining a larger amount of a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, and 19.4 ± 0.2° 20, preferably a crystalline form of a compound according to Formula (1) having an X-ray powder diffraction pattern comprising characteristic peaks at 6.5, 10.7, 15.6, 19.4, 21.5, and 22.8 ± 0.2° 20, as measured by X-ray powder diffraction using an X-ray wavelength of 1.5406 A (Pattern L).

35. The method according to claim 34, wherein the suspension is equilibrated at a temperature of about 50°C.

36. A pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of the TLR4 agonist as defined in any one of claims 1 to 10, or of a crystalline form of the TLR4 agonist as obtained by a method according to any one ofclaims 12-17, 19-22 and 24-26, or of a crystalline form of the TLR4 agonist as prepared by a method according to any one of claims 28 to 35, and a pharmaceutical acceptable carrier and / or expedient.

37. The pharmaceutical composition according to claim 36, wherein the pharmaceutical composition comprises at least one further active ingredient.

38. The pharmaceutical composition according to claim 37, wherein the at least one further active ingredient is selected from the group consisting of a further adjuvant, such as a TLR7 agonist, an immunotherapeutic drug, such as a checkpoint inhibitor, an antigen and / or a virostatic agent.

39. The pharmaceutical composition according to any one of claims 36 to 38, for use as a vaccine.

40. The pharmaceutical composition according to according to any one of claims 36 to 38, for use as an anti-cancer medicament.

41. The pharmaceutical composition according to according to any one of claims 36 to 40, wherein the TLR4 agonist and optionally further active ingredients are formulated in liposomes.

42. A crystalline form of the TLR4 agonist as defined in any one of claims 1 to 10, or a crystalline form of the TLR4 agonist as obtained by a method according to any one of claims 11-17, 19-22 and 24-26, or a crystalline form of the TLR4 agonist as prepared by a method according to any one of claims 28 to 35, or of the pharmaceutical composition according to any one of claims 36 to 41 for use as an adjuvant or anticancer drug.

43. A method for the synthesis of a starting material used in a method according to any one of claims 11-17, 19-22, 24-26, and 28-35, wherein the synthesis method comprises the steps of:i) reacting ethyl-3-amino-5-bromo-1 H-indole-2 -carboxylate according to Formula (2):NH2 / B'VT4 N 00H(2)with methyl iodide to obtain ethyl-1 -methyl-3-amino-5-bromo-1 H-indole-2 - carboxylate according to Formula (3):(3);ii) reacting ethyl-1 -methyl-3-amino-5-bromo-1 H-indole-2 -carboxylate obtained in step i) with 2-furanboronic acid pinacol ester to obtain ethyl-1 -methyl-3-amino- 5-(2-furyl)-1 H-indole-2 -carboxylate according to Formula (4):(4);iii) reacting ethyl-1 -methyl-3-amino-5-(2-furyl)-1 H-indole-2-carboxylate obtained in step ii) with phenyl isothiocyanate to obtain ethyl-1 -methyl-5-(2-furyl)-3-(3- phenylthioureido)amino-1 H-indole-2 -carboxylate according to Formula (5):iv) reacting ethyl-1 -methyl-5-(2-furyl)-3-(3-phenylthioureido)amino-1 H-indole-2- carboxylate obtained in step iii) with sodium ethoxide to obtain 8-(2-furyl)-5- methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4-b]-indol-4(5H)-one according to Formula (6):(6); andv) reacting 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4- b]-indol-4(5H)-one obtained in step iv) with 2-chloro-N-cyclohexylacetamide to obtain 2-((8-(2-furyl)-5-methyl-4-oxo-3-phenyl-4,5-di hydro-3 H-pyrimido-(5, 4- b)indol-2-yl)thio)-N-cyclohexylacetamide according to Formula (1):

44. A synthesis method according to claim 43, wherein the reaction in step i) is conducted in dimethylformamide in the presence of sodium hydride.

45. A synthesis method according to claim 43 or 44, wherein the reaction in step ii) is conducted in dimethoxyethane in the presence of sodium carbonate and water.

46. A synthesis method according to according to any one of claims 43 to 45, wherein the reaction in step ii) is conducted in the presence of a catalyst, such as [1,1 bis(diphenylphosphino)ferrocene] dichloropalladium (II).

47. A synthesis method according to any one of claims 43 to 46, wherein step iii) is conducted in acetonitrile.

48. A synthesis method according to any one of claims 43 to 47, wherein step (iv) is conducted in EtOH.

49. A synthesis method according to any one of claims 43 to 48, wherein step (v) is conducted in dimethylformamide in the presence of triethylamine.

50. A compound, ethyl-1-methyl-3-amino-5-bromo-1 H-indole-2-carboxylate, according to formula (2):(3).

51. A compound, ethyl 1 -methyl-3-amino-5-(2-furyl)-1 H-indole-2-carboxylate, according to formula (4):(4).

52. A compound, ethyl 1-methyl-5-(2 -fury l)-3-(3-phenylthioureido)ami no-1 H-indole-2- carboxylate, according to formula (5):

53. A compound, 8-(2-furyl)-5-methyl-3-phenyl-2-thioxo-2,3-dihydro-1 H-pyrimido[5,4- b]-indol-4(5H)-one, according to formula (6):O

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